Transglutaminase-mediated conjugation

ABSTRACT

The present disclosure provides for antibody-oligonucleotide conjugates, methods of preparation thereof, and methods of use thereof. Also provided are related compounds, compositions and kits.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and benefit of U.S. ProvisionalPatent Application No. 62/983,463, filed on Feb. 28, 2020, and U.S.Provisional Patent Application 63/110,854, filed on Nov. 6, 2020, thedisclosure of each of which is hereby incorporated by reference in itsentirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 186492000200SEQLIST.TXT,date recorded: Feb. 26, 2021, size: 170 KB).

FIELD OF INVENTION

The present disclosure relates generally to methods for conjugating anoligonucleotide and a polypeptide and related compounds, compositionsand kits.

BACKGROUND

Pathogen-associated molecular patterns (PAMPs) are molecules associatedwith various pathogens and are recognized by toll-like receptors (TLRs)and other pattern recognition receptors (PRRs) activating innate immuneresponses. The ability of PAMPs to recruit immune system in the absenceof pathogens provides a strategy for treating a variety of diseasesinvolving cell destruction (e.g., anticancer therapy) through the use ofinnate immune system response. One class of PAMPs that has beeninvestigated for a variety of therapeutic applications isimmunostimulating polynucleotides, such as unmethylated cytosine-guaninedinucleotide (CpG)-containing oligodeoxynucleotides (CpG ODNs) (e.g.,agatolimod). It is thought that CpG ODNs mediate TLR9 dimerization inimmune cells (e.g., B cells, monocytes and plasmacytoid dendritic cells(pDCs)) to upregulate cytokines (e.g., type I interferon andinterleukins), thereby activating natural killer cells.

Toll-like receptor 9 (TLR9), also designated as CD289, is an importantreceptor expressed in immune system cells including dendritic cells(DCs), B lymphocytes, macrophages, natural killer cells, and otherantigen presenting cells. TLR9 activation triggers intracellularsignaling cascades, leading to activation, maturation, proliferation andcytokine productions in these immune cells, thus bridges the innate andadaptive immunity. Martinez-Campos et al., Viral Immunol. 2016, 30,98-105; Notley et al., Sci. Rep. 2017, 7, 42204. Natural TLR-9 agonistsinclude unmethylated cytosine-guanine dinucleotide (CpG)-containingoligodeoxynucleotides (CpG ODNs).

CpG ODNs may include, for example, oligodeoxynucleotides having poly-Gtails with phosphorothioate backbones at 3′- and 5′-termini and acentral palindromic sequence including a phosphate backbone and a CpGwithin its central palindrome sequence, or oligodeoxynucleotides havinga fully phosphorothioate backbone, and a sequence at the 5′ end for TLR9activation, or oligodeoxynucleotides having a fully phosphorothioatebackbone with a 3′-end sequence enabling formation of a duplex. However,CpG ODNs are often susceptible to degradation in serum and thuspharmacokinetics of CpG ODNs may be one of the limiting factors in theirdevelopment as therapeutics. Also CpG ODNs often exhibit uneven tissuedistribution in vivo, with primary sites of accumulation being in liver,kidney, and spleen. Such distribution can elicit off-target activity andlocal toxicity associated with PAMPs.

One solution is to conjugate the immunomodulating polynucleotides (e.g.,CpG ODNs) with a targeting moiety for specifically targeted tissues orcells to overcome the uneven distribution of the polynucleotide. See US2018/0312536. Particularly, transglutaminase-mediated reaction can beused to conjugate a polypeptide targeting moiety containing a glutamineresidue with a CpG ODN containing a primary amine group. Microbialtransglutaminase (mTG) is from the species Streptomyces mobaraensis. ThemTG catalyzes under pH-controlled aqueous conditions (includingphysiological conditions) a transamidation reaction between a ‘reactive’glutamine of a protein and a ‘reactive’ lysine residue whereas thelatter can also be a simple, low molecular weight primary amine such asa 5-aminopentyl group. For an endogenous glutamine on a protein to berecognized as an mTG-substrate two criteria seem important: 1) thepresence of hydrophobic amino acids in the peptide sequence adjacent tothe glutamine residue and 2) the positioning of the glutamine on a loopwith local chain flexibility enhancing reactivity toward mTG.

Although conjugation of these immunomodulating polynucleotides may leadto improved stability and distribution, there remains a need forimmunomodulating polynucleotides with improved stability and selectivitywith or without conjugation to targeting moieties, and methods forpreparing them.

BRIEF SUMMARY

In one aspect, provided herein is a conjugate comprising an antibody orantigen-binding fragment thereof and one or more immunomodulatingoligonucleotides (P), wherein each immunomodulating oligonucleotide islinked via an amide bond to a glutamine residue (Q) of the antibody orfragment and a linker (L) as shown in Formula (A):

wherein

indicates the point of attachment of each Q to the antibody orantigen-binding fragment thereof (Ab). In some embodiments, theglutamine residue is part of a Q-tag peptide linked to the antibody,e.g., to the C-terminus of the antibody Fc region. In some embodiments,the glutamine residue is part of the antibody (e.g., part of the Fcregion, such as residue Q295). In some embodiments, the antibody furthercomprises an N297A mutation. Exemplary Q-tag peptide sequences areprovided, e.g., in Table 3. Exemplary immunomodulating oligonucleotidesare described herein and provided, e.g., in Tables 2 & 9-12. In someembodiments, 1-4 immunomodulating oligonucleotides are conjugated to theantibody. Exemplary linkers (L) are described herein. In someembodiments, the linker comprises a polyethylene glycol moiety.

In one aspect, provided herein is a conjugate comprising an antibody orantigen-binding fragment thereof and one or more immunomodulatingoligonucleotides (P), wherein the antibody or antigen-binding fragmentis linked to one or more Q-tag peptides (Q) that comprise the amino acidsequence RPQGF (SEQ ID NO:47), wherein each immunomodulatingoligonucleotide is linked to a Q-tag peptide via an amide bond with theglutamine residue of the Q-tag peptide and a linker (L) as shown inFormula (A):

wherein

indicates the point of attachment of each Q to the antibody orantigen-binding fragment thereof (Ab).

In some embodiments, the antibody is linked to 2 Q-tag peptides, and oneof the Q-tag peptides is linked to an immunomodulating oligonucleotide.In some embodiments, the antibody is linked to 2 Q-tag peptides, andeach of the 2 Q-tag peptides is linked to an immunomodulatingoligonucleotide. In some embodiments, the antibody or fragment thereofis a monoclonal antibody or fragment thereof. In an additionalembodiment of the present aspect, the antibody or fragment thereof is aFab, F(ab′)2, Fab′-SH, Fv, scFv, single domain, single heavy chain, orsingle light chain antibody or antibody fragment. In yet anotherembodiment of this aspect which may be combined with any of thepreceding embodiments, the antibody or fragment thereof is a humanized,human, or chimeric antibody or fragment thereof. In still furtherembodiments of the present aspect, the antibody or fragment thereofspecifically binds a tumor associated antigen.

In still further embodiments of the present aspect, the antibody orfragment thereof specifically binds human CD22. In still yet anotherembodiment of the present aspect, the antibody comprises a heavy chainvariable (VH) domain and a light chain variable (VL) domain, wherein theVH domain comprises CDR-H1, CDR-H2, and CDR-H3 sequences from a VHdomain sequence selected from the group consisting of:

(SEQ ID NO: 64) EVQLVESGGGLVQPGGSLRLSCAASGFAFSIYDMSWVRQAPGKGLEWVAYISSGGGTTYYPDTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHSGYGTHWGVLFAYWGRGTLVTVSS, (SEQ ID NO: 65)QVQLLESGGGVVQPGGSLRLSCAASGFAFSIYDMNWVRQAPGKGLEWVSAISSGGGTTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHSGYGTHWGVLFAYWGRGTLVTVSS, (SEQ ID NO: 66)EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYEMNWVRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHSGYGTHWGVLFAYWGRGTLVTVSS, and (SEQ ID NO: 67)QVQLQESGPGLVKPSDTLSLTCTVSGFAFSIYDMSWIRQPPGKGLEWIAYISSGGGTTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARHSGYGTHWGVLFAYWGRGTLVTVSS.In still et another embodiment of the present aspect, the antibodycomprises a heavy chain variable (VH) domain and a light chain variable(VL) domain, wherein the VH domain comprises CDR-H1, CDR-H2, and CDR-H3sequences from a VH domain shown in Table 8. In still yet anotherembodiment of the present aspect, the antibody comprises a heavy chainvariable (VH) domain and a light chain variable (VL) domain, wherein theVH domain comprises a CDR-H1 comprising the sequence of SEQ ID NO:113, aCDR-H2 comprising the sequence of SEQ ID NO:115, and a CDR-H3 comprisingthe sequence of SEQ ID NO:116; or a CDR-H1 comprising the sequence ofSEQ ID NO:114, a CDR-H2 comprising the sequence of SEQ ID NO:189, and aCDR-H3 comprising the sequence of SEQ ID NO:116. In still yet anotherembodiment of the present aspect, the antibody comprises a heavy chainvariable (VH) domain and a light chain variable (VL) domain, wherein theVH domain comprises a CDR-H1 comprising the sequence of SEQ ID NO:113, aCDR-H2 comprising the sequence of SEQ ID NO:115, and a CDR-H3 comprisingthe sequence of SEQ ID NO:116, and the VL domain comprises a CDR-L1comprising the sequence of SEQ ID NO:117, a CDR-L2 comprising thesequence of SEQ ID NO:119, and a CDR-L3 comprising the sequence of SEQID NO:120. In yet another embodiment of the present aspect, the antibodycomprises a heavy chain variable (VH) domain and a light chain variable(VL) domain, and wherein the VH domain comprises an amino acid sequenceselected from the group consisting of

(SEQ ID NO: 64) EVQLVESGGGLVQPGGSLRLSCAASGFAFSIYDMSWVRQAPGKGLEWVAYISSGGGTTYYPDTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHSGYGTHWGVLFAYWGRGTLVTVSS, (SEQ ID NO: 65)QVQLLESGGGVVQPGGSLRLSCAASGFAFSIYDMNWVRQAPGKGLEWVSAISSGGGTTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHSGYGTHWGVLFAYWGRGTLVTVSS, (SEQ ID NO: 66)EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYEMNWVRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHSGYGTHWGVLFAYWGRGTLVTVSS, and (SEQ ID NO: 67)QVQLQESGPGLVKPSDTLSLTCTVSGFAFSIYDMSWIRQPPGKGLEWIAYISSGGGTTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARHSGYGTHWGVLFAYWGRGTLVTVSS.In yet another embodiment of the present aspect, the antibody comprisesa heavy chain variable (VH) domain and a light chain variable (VL)domain, wherein the VH domain comprises the sequence of SEQ ID NO:65,and the VL domain comprises the sequence of SEQ ID NO:87. In still yetanother embodiment of the present aspect, the antibody comprises a heavychain variable (VH) domain and a light chain variable (VL) domain,wherein the VL domain comprises CDR-L1, CDR-L2, and CDR-L3 sequencesfrom a VL domain sequence selected from the group consisting of:

(SEQ ID NO: 68) DIQMTQSPSSLSASVGDRVTITCRASQDIHGYLNWYQQKPGKAPKLLIYYTSILHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQGNTLPWTFGQ GTKLEIK,(SEQ ID NO: 69) DIQMTQSPSSVSASVGDRVTITCRASQDIHGYLAWYQQKPGKAPKLLIYYTSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNTLPWTFGQ GTKLEIK,(SEQ ID NO: 70) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNTLPWTFGQ GTKLEIK,(SEQ ID NO: 71) EIVLTQSPATLSLSPGERATLSCRASQDIHGYLNWYQQKPGQAPRLLIYYTSILHSGIPARFSGSGPGTDFTLTISSLEPEDFAVYYCQQGNTLPWTFGG GTKLEIK, and(SEQ ID NO: 72) DIVMTQTPLSLSVTPGQPASISCRASQDIFIGYLNWYQQKPGQSPQLLIYYTSILHSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCQQGNTLPWTFG GGTKLEIK.In still yet another embodiment of the present aspect, the antibodycomprises a heavy chain variable (VH) domain and a light chain variable(VL) domain, wherein the VL domain comprises CDR-L1, CDR-L2, and CDR-L3sequences from a VL domain sequence selected from the group consistingof SEQ ID Nos: 68-91. In still yet another embodiment of the presentaspect, the antibody comprises a heavy chain variable (VH) domain and alight chain variable (VL) domain, wherein the VL domain comprisesCDR-L1, CDR-L2, and CDR-L3 sequences from a VL domain shown in Table 8.In still yet another embodiment of the present aspect, the antibodycomprises a heavy chain variable (VH) domain and a light chain variable(VL) domain, wherein the VL domain comprises a CDR-L1 comprising thesequence of SEQ ID NO:117, a CDR-L2 comprising the sequence of SEQ IDNO:119, and a CDR-L3 comprising the sequence of SEQ ID NO:120; or aCDR-L1 comprising the sequence of SEQ ID NO:118, a CDR-L2 comprising thesequence of SEQ ID NO:177, and a CDR-L3 comprising the sequence of SEQID NO:120. In still further embodiments of the present aspect, the VLdomain further comprises an amino acid substitution at residue N92. Incertain embodiments wherein the VL domain comprises an amino acidsubstitution at residue N92, the amino acid substitution at residue N92is selected from the group consisting of N92A, N92L and N92S. In stillfurther embodiments of the present aspect, the antibody comprises aheavy chain variable (VH) domain and a light chain variable (VL) domain,wherein the VL domain an amino acid sequence selected from the groupconsisting ofDIQMTQSPSSLSASVGDRVTITCRASQDIHGYLNWYQQKPGKAPKLLIYYTSILHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQGNTLPWTFGQGTKLEIK (SEQ ID NO: 68),DIQMTQSPSSVSASVGDRVTITCRASQDIHGYLAWYQQKPGKAPKLLIYYTSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKLEIK (SEQ ID NO: 69),DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKLEIK (SEQ ID NO: 70),EIVLTQSPATLSLSPGERATLSCRASQDIHGYLNWYQQKPGQAPRLLIYYTSILHSGIPARFSGSGPGTDFTLTISSLEPEDFAVYYCQQGNTLPWTFGGGTKLEIK (SEQ ID NO: 71), andDIVMTQTPLSLSVTPGQPASISCRASQDIHGYLNWYQQKPGQSPQLLIYYTSILHSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCQQGNTLPWTFGGGTKLEIK (SEQ ID NO: 72). In stillfurther embodiments of the present aspect, the antibody comprises aheavy chain variable (VH) domain and a light chain variable (VL) domain,wherein the VH domain comprises the amino acid sequenceQVQLLESGGGVVQPGGSLRLSCAASGFAFSIYDMNWVRQAPGKGLEWVSAISSGGGTTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHSGYGTHWGVLFAYWG RGTLVTVSS (SEQID NO: 65), and wherein the VL domain comprises an amino acid sequenceselected from the group consisting of

(SEQ ID NO: 68) DIQMTQSPSSLSASVGDRVTITCRASQDIHGYLNWYQQKPGKAPKLLIYYTSILHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQGNTLPWTFGQ GTKLEIK,(SEQ ID NO: 73) DIQMTQSPSSLSASVGDRVTITCRASQDIHGYLNWYQQKPGKAPKLLIYYTSILHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQGATLPWTFGQ GTKLEIK,(SEQ ID NO: 82) DIQMTQSPSSLSASVGDRVTITCRASQDIHGYLNWYQQKPGKAPKLLIYYTSILHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQGLTLPWTFGQ GTKLEIK, and(SEQ ID NO: 87) DIQMTQSPSSLSASVGDRVTITCRASQDIHGYLNWYQQKPGKAPKLLIYYTSILHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQGSTLPWTFGQ GTKLEIK.

In still further embodiments, the antibody or fragment thereofspecifically binds human Her2. In still yet another embodiment of thepresent aspect, the antibody comprises a heavy chain variable (VH)domain and a light chain variable (VL) domain, wherein the VH domaincomprises CDR-H1, CDR-H2, and CDR-H3 sequences from the VH domainsequence EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGT LVTVSS (SEQ IDNO:168) and/or wherein the VL domain comprises CDR-L1, CDR-L2, andCDR-L3 sequences from the VL domain sequence

(SEQ ID NO: 169) DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQ GTKVEIK.In still yet another embodiment of the present aspect, the antibodycomprises a heavy chain variable (VH) domain and a light chain variable(VL) domain, wherein the VH domain comprises the sequenceEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGT LVTVSS (SEQ IDNO:168) and/or wherein the VL domain comprises the sequenceDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO:169).In still yet another embodiment of the present aspect, the antibodycomprises a heavy chain variable (VH) domain and a light chain variable(VL) domain, wherein the VH domain comprises CDR-H1, CDR-H2, and CDR-H3sequences from the VH domain sequenceEVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLV TVSS (SEQ IDNO:170) and/or wherein the VL domain comprises CDR-L1, CDR-L2, andCDR-L3 sequences from the VL domain sequenceDIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIK (SEQ ID NO:171). Instill yet another embodiment of the present aspect, the antibodycomprises a heavy chain variable (VH) domain and a light chain variable(VL) domain, wherein the VH domain comprises the sequenceEVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLV TVSS (SEQ IDNO:170) and/or wherein the VL domain comprises the sequenceDIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIK (SEQ ID NO:171). In someembodiments, the anti-Her2 antibody is trastuzumab or pertuzumab

In still yet another embodiments of the present aspect that can becombined with any of the preceding aspects, the antibody comprises an Fcregion. In certain embodiments, the Fc region is a human Fc regionselected from the group consisting of an IgG1 Fc region, an IgG2 Fcregion, and an IgG4 Fc region. In some embodiments, the Fc region is awild-type human IgG1, IgG2, or IgG4 Fc region. In some embodiments, theFc region is a human Fc region comprising one or more amino acidsubstitutions that reduce one or more effector functions, as comparedwith the effector function(s) of a human Fc region that lacks the aminoacid substitution(s). In still further embodiments, the Fc region is:(a) a human IgG1 Fc region comprising L234A, L235A, and/or G237Asubstitutions, amino acid position numbering according to EU index; (b)a human IgG2 Fc region comprising A330S and/or P331S substitutions,amino acid position numbering according to EU index; or (c) a human IgG4Fc region comprising S228P and/or L235E substitutions, amino acidposition numbering according to EU index. In still yet anotherembodiment, the Fc region further comprises an N297A substitution, aminoacid position numbering according to EU index. In some embodiments, theantibody comprises an antibody heavy chain constant domain comprising anamino acid sequence selected from the group consisting of SEQ IDNos:92-107 and 178. In certain embodiments wherein the Fc regioncomprises an N297A substitution, the conjugate further comprises animmunomodulating oligonucleotide P attached to the Q295 residue of theFc region as shown in the following formula

wherein L is a linker moiety connected to Q295 via an amide bond. In yetother embodiments, the Fc region further comprises a D265A substitution,amino acid position numbering according to EU index. In someembodiments, the conjugate binds to human CD22 expressed on the surfaceof a B cell. In some embodiments, the conjugate induces activation ofTLR9.

In some embodiments of the present aspect, the antibody comprises ahuman lambda light chain. In other embodiments of the present aspect,the antibody comprises a human kappa light chain. In some embodiments,the antibody comprises an antibody light chain constant domaincomprising an amino acid sequence selected from the group consisting ofSEQ ID Nos:108-110. In still further embodiments which may be combinedwith any preceding embodiments of the present aspect, at least one Q-tagis attached to the heavy chain of the antibody. In certain embodiments,at least one Q-tag is fused to the C-terminus of the heavy chain of theantibody. In other embodiments, at least one Q-tag is attached to thelight chain of the antibody. In still yet other embodiments, 1 or 2Q-tags is/are linked to the antibody or antigen-binding fragment. Instill further embodiments, the conjugate has a DAR of 1. In stillfurther embodiments, the conjugate has a DAR of 2.

In still further embodiments of the present aspect, eachimmunomodulating oligonucleotide P is independently

whereinb and c are each independently an integer from 1 to 25; with the provisothat the sum of b and c is at least 5;

indicates the point of attachment of the immunomodulatingoligonucleotide P to the rest of the conjugate;X^(5′) is a 5′ terminal nucleoside having the structure

X^(3′) is a 3′ terminal nucleoside having the structure

Y^(PTE) is an internucleoside phosphotriester having the structure

wherein * indicates the points of attachment to the rest of theoligonucleotide and

indicates the point of attachment to the linker L, or, if L is absent,

indicates the point of attachment to the Q tag peptide Q at theglutamine residue via an amide bond;Y^(3′) is a terminal phosphotriester having the structure

each X^(N) is independently a nucleoside having the structure

each Y^(N) is independently an internucleoside linker having thestructure

wherein each B^(N) is independently a modified or unmodified nucleobase;each R^(N) is independently —H or —O—C₁₋₄-alkyl, wherein the C₁₋₄-alkylof the —O—C₁₋₄-alkyl is further optionally substituted by—O—C₁-C₄-alkyl;B^(5′) and B^(3′) are independently a modified or unmodified nucleobase;R^(5′) and R^(3′) are independently —H or —O—C₁-C₄-alkyl, wherein theC₁₋₄-alkyl of the —O—C₁₋₄-alkyl is further optionally substituted by—O—C₁-C₄-alkyl;each T₁ is independently O or S;each T₂ is independently O⁻ or S⁻; andT₃ is a group comprising an oligoethylene glycol moiety; andR¹ is C₁₋₄-alkylene-hydroxy.

In certain embodiments of the present aspect, b is 3. In additionalembodiments of the present aspect, (i) P comprises at least one modifiednucleoside X^(N); (ii) P has at least one modified internucleosidelinker Y^(N), wherein at least one of T¹ or T² is S; or (iii) both (i)and (ii). In some embodiments, P has at least one phosphorodithioate orphosphorothioate internucleoside linker. In certain embodiments, Pcomprises 0, 1, 2 or 3 phosphorodithioate internucleoside linkers. Instill further embodiments, P comprises a modified nucleoside selectedfrom the group consisting of 2′-O-alkyl nucleoside, 2′-O-alkoxyalkylnucleoside, 2′-deoxynucleoside and ribonucleoside. In certainembodiments, the modified nucleoside is selected from the groupconsisting of 5-bromo-2′-O-methyluridine, 5-bromo-2′-deoxyuridine,2′-O-methyluridine, 2′-deoxyuridine, 2′-O-methylthymidine,2′-O-methylcytidine, 2′-O-(2-methoxyethyl)thymidine and8-oxo-7,8-dihydro-2′-deoxyguanosine. In yet other embodiments, Y^(3′) orthe Y^(N) at the 3′ position of X^(5′) comprises an unsubstituted orsubstituted phosphorothioate.

In yet other embodiments of the present aspect, Y^(PTE) is:

wherein Z is O or S; d is an integer from 0 to 95; the two

on the right side of the structure indicate the points of attachment tothe adjacent nucleosides X^(N) in the oligonucleotide P, and the

on the left side of the structure indicates the point of attachment tothe linker L. In other embodiments, Y^(PTE) is:

wherein Z is O or S; d is an integer from 0 to 95; the two

on the right side of the structure indicate the points of attachment tothe adjacent nucleosides X^(N) in the oligonucleotide P, and the one fon the left side of the structure indicates the point of attachment tothe linker L. In certain embodiments, Z is S. In still furtherembodiments, d is an integer from 1 to 25. In additional embodimentswhich may be combined with any of the preceding embodiments, the linkerL comprises a polyethylene glycol moiety. In yet further embodiments,the linker L is

wherein m is an integer ranging from about 0 to about 50, and wherein

indicates the point of attachment to Y^(PTE), and

indicates the point of attachment to the rest of the conjugate. In stillfurther embodiments, P comprises one or more CpG sites. In still anotherembodiment, P comprises at least 3 CpG sites. In yet other embodiments,each P independently comprises an oligonucleotide sequence selected fromthe group consisting of the oligonucleotides of Table 9 and Table 10.

In some embodiments, the conjugate comprises one or more, two or more,three or more, four or more, five or more, or ten or more Q-tagpeptides. In some embodiments, the conjugate comprises two Q-tagpeptides. In some embodiments, the conjugate comprises one or more, twoor more, three or more, four or more, five or more, or ten or moreimmunomodulating oligonucleotides. In some embodiments, the conjugatecomprises one immunomodulating oligonucleotide. In certain embodiments,the antibody is linked to 2 Q-tag peptides, and wherein one of the Q-tagpeptides is linked to an immunomodulating oligonucleotide. In someembodiments, the antibody comprises two antibody light chains, twoantibody heavy chains, and two Q-tag peptides; wherein each of the Q-tagpeptides is linked to the C-terminus of one of the antibody heavychains; and wherein one of the Q-tag peptides is linked to animmunomodulating oligonucleotide (P) via an amide bond with theglutamine residue of the Q-tag peptide and linker (L), e.g., as shown inFIG. 16 .

In yet another aspect, the present disclosure provides a conjugate thatcomprises an antibody or antigen-binding fragment thereof (Ab) and oneor more immunomodulating oligonucleotides (P), wherein the antibody orantigen-binding fragment is linked to one or more Q-tag peptides (Q)comprising at least one glutamine residue, wherein each immunomodulatingoligonucleotide is linked to a Q-tag peptide via an amide bond with theglutamine residue of the Q-tag peptide and a linker (L) as shown inFormula (A),

wherein:

indicates the point of attachment of each Q to the antibody orantigen-binding fragment thereof (Ab);each Q is independently a Q-tag peptide sequence having at least oneglutamine residue;each L is independently a bond or a linker moiety connected to Q via anamide bond with the glutamine residue; andeach P is independently an immunomodulating oligonucleotide of havingthe structure

wherein

and

indicate the points of attachment within the oligonucleotide;

each T¹ is independently O or S;each T² is S⁻;T³ is a group

wherein

indicates the point of attachment to Land wherein

indicates the point of attachment to the rest of the oligonucleotide;

Z is O or S;

U^(5′) is —H or halogen;R^(5′) is —H or methoxy;R^(c1) is —H or methoxy;R^(g1), R^(g2), R^(g3), and R^(g4) are H;R^(3′) is methoxy;R¹ is —(CH₂)₃—OH;R² is —H or methyl; andn is an integer from 0 to 2.In still further embodiments of the present aspect, the antibody orfragment thereof specifically binds a tumor associated antigen.

In yet another aspect, the present disclosure provides a conjugate thatcomprises an antibody or antigen-binding fragment thereof (Ab) and oneor more immunomodulating oligonucleotides (P), wherein the antibody orantigen-binding fragment is linked to one or more Q-tag peptides (Q)comprising at least one glutamine residue, wherein each immunomodulatingoligonucleotide is linked to a Q-tag peptide via an amide bond with theglutamine residue of the Q-tag peptide and a linker (L) as shown inFormula (A),

wherein:

indicates the point of attachment of each Q to the antibody orantigen-binding fragment thereof (Ab);each Q is independently a Q-tag peptide sequence having at least oneglutamine residue;each L is independently a bond or a linker moiety connected to Q via anamide bond with the glutamine residue; andeach P is independently an immunomodulating oligonucleotide of havingthe structure

wherein

and

indicate the points of attachment within the oligonucleotide;

each T¹ is independently O or S;each T² is S⁻;T³ is a group

wherein

indicates the point of attachment to L and wherein

indicates the point of attachment to the rest of the oligonucleotide;

Z is O or S;

R^(5′) is —H or methoxy;R^(c1) is —H or methoxy;R^(g1), R^(g2), R^(g3), and R^(g4) are H;R^(3′) is methoxy;R¹ is —(CH₂)₃—OH;R² is —H or methyl; andn is an integer from 0 to 2.In still further embodiments of the present aspect, the antibody orfragment thereof specifically binds a tumor associated antigen.

In another aspect, provided herein is a conjugate comprising an antibodyor antigen-binding fragment thereof (Ab) and one or moreimmunomodulating oligonucleotides (P), wherein the antibody orantigen-binding fragment is linked to one or more Q-tag peptides (Q)comprising a Q-tag peptide sequence RPQGF (SEQ ID NO:47), and whereineach immunomodulating oligonucleotide is linked to a Q-tag peptide viaan amide bond with the glutamine residue of the Q-tag peptide and alinker (L) as shown in Formula (A)

wherein:

indicates the point of attachment of each Q to the antibody orantigen-binding fragment thereof (Ab)

each Q independently comprises a Q-tag peptide sequence RPQGF (SEQ IDNO:47);

each L is independently a bond or a linker moiety

wherein m is an integer ranging from about 0 to about 50, and wherein

indicates the point of attachment to P, and

indicates the point of attachment to the rest of the conjugate connectedto Q via an amide bond with the glutamine residue; and

each P is independently an immunomodulating oligonucleotide having thestructure

wherein

and

indicate the points of attachment within the oligonucleotide;

each T¹ is independently O or S;

each T² is S⁻;

T³ is a group

wherein

indicates the point of attachment to L and wherein

indicates the point of attachment to the rest of the oligonucleotide;

Z is O or S;

U^(5′) is —H or halogen;

R^(5′) is —H or methoxy;

R is —H or methoxy;

R^(g1), R^(g2), R^(g3), and R^(g4) are H;

R^(3′) is methoxy;

R¹ is —(CH₂)₃—OH;

R² is —H or methyl; and

n is an integer from 0 to 2,

wherein Ab is an antibody or antigen-binding fragment thereof that bindsa tumor associated antigen.

In yet another aspect, the present disclosure provides a conjugatecomprising an antibody or antigen-binding fragment thereof (Ab) and oneor more immunomodulating oligonucleotides (P), wherein the antibody orantigen-binding fragment is linked to one or more Q-tag peptides (Q)comprising a Q-tag peptide sequence RPQGF (SEQ ID NO:47), and whereineach immunomodulating oligonucleotide is linked to a Q-tag peptide viaan amide bond with the glutamine residue of the Q-tag peptide and alinker (L) as shown in Formula (A)

wherein:

indicates the point of attachment of each Q to the antibody orantigen-binding fragment thereof (Ab)

each Q independently comprises a Q-tag peptide sequence RPQGF (SEQ IDNO:47);

each L is independently a bond or a linker moiety

wherein m is an integer ranging from about 0 to about 50, and wherein

indicates the point of attachment to P, and

indicates the point of attachment to the rest of the conjugate connectedto Q via an amide bond with the glutamine residue; and

each P is independently an immunomodulating oligonucleotide having thestructure

wherein

and

indicate the points of attachment within the oligonucleotide; wherein Abis an antibody or antigen-binding fragment thereof that binds a tumorassociated antigen.

In some embodiments, the tumor associated antigen is expressed by acancer cell. In some embodiments, the tumor associated antigen isexpressed by a cancer-associated stromal cell. In some embodiments, thetumor associated antigen is selected from the group consisting of CD19,CD20, CD22, CD25, CD30, CD33, CD38, CD40, CD44, CD45R (B220), CD49,CD52, CD56, CD70, CD74, CD79a, CD79b, CD93, CD123, CD138, CD163, CD205,CD206, CD274, CD303, and CD304, folate receptor alpha, folate receptorbeta, mesothelin, PSMA, Her-2, EGFR, CLDN18.2, 5T4, CD47, nectin 4,transferrin receptor, integrin, cripto, EphA2, AGS-5, AGS-16, CanAg,EpCAM, IL4 receptor, IL2 receptor, Lewis Y, GPNMB, DLL3, GCC, GPA33,tissue factor (TF), and Trop2. In some embodiments, the cancer is breastcancer, colorectal cancer, lung cancer, head and neck cancer, melanoma,lymphoma, or leukemia.

In some embodiments of the present aspect, the antibody or fragmentthereof is a monoclonal antibody or fragment thereof. In someembodiments, the antibody is linked to 2 Q-tag peptides, and wherein oneof the Q-tag peptides is linked to an immunomodulating oligonucleotide.In additional embodiments of the present aspect, the antibody orfragment thereof is a Fab, F(ab′)2, Fab′-SH, Fv, scFv, single domain,single heavy chain, or single light chain antibody or antibody fragment.In yet other embodiments of the present aspect, the antibody or fragmentthereof is a humanized, human, or chimeric antibody or fragment thereof.

In still further embodiments, the antibody or fragment thereofspecifically binds human CD22. In still yet another embodiment of thepresent aspect, the antibody comprises a heavy chain variable (VH)domain and a light chain variable (VL) domain, wherein the VH domaincomprises CDR-H1, CDR-H2, and CDR-H3 sequences from a VH domain sequenceselected from the group consisting of:

(SEQ ID NO: 64) EVQLVESGGGLVQPGGSLRLSCAASGFAFSIYDMSWVRQAPGKGLEWVAYISSGGGTTYYPDTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHSGYGTHWGVLFAYWGRGTLVTVSS, (SEQ ID NO: 65)QVQLLESGGGVVQPGGSLRLSCAASGFAFSIYDMNWVRQAPGKGLEWVSAISSGGGTTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHSGYGTHWGVLFAYWGRGTLVTVSS, (SEQ ID NO: 66)EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYEMNWVRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHSGYGTHWGVLFAYWGRGTLVTVSS, and (SEQ ID NO: 67)QVQLQESGPGLVKPSDTLSLTCTVSGFAFSIYDMSWIRQPPGKGLEWIAYISSGGGTTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARHSGYGTHWGVLFAYWGRGTLVTVSS.In still yet another embodiment of the present aspect, the antibodycomprises a heavy chain variable (VH) domain and a light chain variable(VL) domain, wherein the VH domain comprises CDR-H1, CDR-H2, and CDR-H3sequences from a VH domain shown in Table 8. In yet another embodimentof the present aspect, the antibody comprises a heavy chain variable(VH) domain and a light chain variable (VL) domain, and wherein the VHdomain comprises an amino acid sequence selected from the groupconsisting of EVQLVESGGGLVQPGGSLRLSCAASGFAFSIYDMSWVRQAPGKGLEWVAYISSGGGTTYYPDTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHSGYGTHWGVLFAYWGR GTLVTVSS (SEQID NO: 64), QVQLLESGGGVVQPGGSLRLSCAASGFAFSIYDMNWVRQAPGKGLEWVSAISSGGGTTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHSGYGTHWGVLFAYWG RGTLVTVSS (SEQID NO: 65), EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYEMNWVRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHSGYGTHWGVLFAYWGR GTLVTVSS (SEQID NO: 66), andQVQLQESGPGLVKPSDTLSLTCTVSGFAFSIYDMSWIRQPPGKGLEWIAYISSGGGTTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARHSGYGTHWGVLFAYWGRGTL VTVSS (SEQ IDNO: 67). In still yet another embodiment of the present aspect, theantibody comprises a heavy chain variable (VH) domain and a light chainvariable (VL) domain, wherein the VL domain comprises CDR-L1, CDR-L2,and CDR-L3 sequences from a VL domain sequence selected from the groupconsisting of:

(SEQ ID NO: 68) DIQMTQSPSSLSASVGDRVTITCRASQDIHGYLNWYQQKPGKAPKLLIYYTSILHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQGNTLPWTFGQ GTKLEIK,(SEQ ID NO: 69) DIQMTQSPSSVSASVGDRVTITCRASQDIHGYLAWYQQKPGKAPKLLIYYTSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNTLPWTFGQ GTKLEIK,(SEQ ID NO: 70) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNTLPWTFGQ GTKLEIK,(SEQ ID NO: 71) EIVLTQSPATLSLSPGERATLSCRASQDIHGYLNWYQQKPGQAPRLLIYYTSILHSGIPARFSGSGPGTDFTLTISSLEPEDFAVYYCQQGNTLPWTFGG GTKLEIK, and(SEQ ID NO: 72) DIVMTQTPLSLSVTPGQPASISCRASQDIFIGYLNWYQQKPGQSPQLLIYYTSILHSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCQQGNTLPWTFG GGTKLEIK.In still yet another embodiment of the present aspect, the antibodycomprises a heavy chain variable (VH) domain and a light chain variable(VL) domain, wherein the VL domain comprises DR-L1, CDR-L2, and CDR-L3sequences from a VL domain shown in Table 8. In still furtherembodiments of the present aspect, the VL domain further comprises anamino acid substitution at residue N92. In certain embodiments whereinthe VL domain comprises an amino acid substitution at residue N92, theamino acid substitution at residue N92 is selected from the groupconsisting of N92A, N92L and N92S. In still further embodiments of thepresent aspect, the antibody comprises a heavy chain variable (VH)domain and a light chain variable (VL) domain, wherein the VL domain anamino acid sequence selected from the group consisting ofDIQMTQSPSSLSASVGDRVTITCRASQDIHGYLNWYQQKPGKAPKLLIYYTSILHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQGNTLPWTFGQGTKLEIK (SEQ ID NO: 68),DIQMTQSPSSVSASVGDRVTITCRASQDIHGYLAWYQQKPGKAPKLLIYYTSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKLEIK (SEQ ID NO: 69),DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKLEIK (SEQ ID NO: 70),EIVLTQSPATLSLSPGERATLSCRASQDIHGYLNWYQQKPGQAPRLLIYYTSILHSGIPARFSGSGPGTDFTLTISSLEPEDFAVYYCQQGNTLPWTFGGGTKLEIK (SEQ ID NO: 71),DIVMTQTPLSLSVTPGQPASISCRASQDIHGYLNWYQQKPGQSPQLLIYYTSILHSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCQQGNTLPWTFGGGTKLEIK (SEQ ID NO: 72),DIQMTQSPSSLSASVGDRVTITCRASQDIHGYLNWYQQKPGKAPKLLIYYTSILHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQGATLPWTFGQGTKLEIK (SEQ ID NO: 73), andDIQMTQSPSSLSASVGDRVTITCRASQDIHGYLNWYQQKPGKAPKLLIYYTSILHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQGLTLPWTFGQGTKLEIK (SEQ ID NO: 82). Instill further embodiments of the present aspect, the antibody comprisesa heavy chain variable (VH) domain and a light chain variable (VL)domain, wherein the VH domain comprises the amino acid sequenceQVQLLESGGGVVQPGGSLRLSCAASGFAFSIYDMNWVRQAPGKGLEWVSAISSGGGTTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHSGYGTHWGVLFAYWG RGTLVTVSS (SEQID NO: 65), and wherein the VL domain comprises an amino acid sequenceselected from the group consisting ofDIQMTQSPSSLSASVGDRVTITCRASQDIHGYLNWYQQKPGKAPKLLIYYTSILHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQGNTLPWTFGQGTKLEIK (SEQ ID NO: 68),DIQMTQSPSSLSASVGDRVTITCRASQDIHGYLNWYQQKPGKAPKLLIYYTSILHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQGATLPWTFGQGTKLEIK (SEQ ID NO: 73),DIQMTQSPSSLSASVGDRVTITCRASQDIHGYLNWYQQKPGKAPKLLIYYTSILHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQGLTLPWTFGQGTKLEIK (SEQ ID NO: 82), andDIQMTQSPSSLSASVGDRVTITCRASQDIHGYLNWYQQKPGKAPKLLIYYTSILHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQGSTLPWTFGQGTKLEIK (SEQ ID NO: 87).

In still further embodiments, the antibody or fragment thereofspecifically binds human Her2. In still yet another embodiment of thepresent aspect, the antibody comprises a heavy chain variable (VH)domain and a light chain variable (VL) domain, wherein the VH domaincomprises CDR-H1, CDR-H2, and CDR-H3 sequences from the VH domainsequence EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGT LVTVSS (SEQ IDNO:168) and/or wherein the VL domain comprises CDR-L1, CDR-L2, andCDR-L3 sequences from the VL domain sequenceDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO:169). Instill yet another embodiment of the present aspect, the antibodycomprises a heavy chain variable (VH) domain and a light chain variable(VL) domain, wherein the VH domain comprises the sequenceEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGT LVTVSS (SEQ IDNO:168) and/or wherein the VL domain comprises the sequenceDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO:169). Instill yet another embodiment of the present aspect, the antibodycomprises a heavy chain variable (VH) domain and a light chain variable(VL) domain, wherein the VH domain comprises CDR-H1, CDR-H2, and CDR-H3sequences from the VH domain sequenceEVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLV TVSS (SEQ IDNO:170) and/or wherein the VL domain comprises CDR-L1, CDR-L2, andCDR-L3 sequences from the VL domain sequenceDIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIK (SEQ ID NO:171). Instill yet another embodiment of the present aspect, the antibodycomprises a heavy chain variable (VH) domain and a light chain variable(VL) domain, wherein the VH domain comprises the sequenceEVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLV TVSS (SEQ IDNO:170) and/or wherein the VL domain comprises the sequenceDIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIK (SEQ ID NO:171). In someembodiments, the anti-Her2 antibody is trastuzumab or pertuzumab.

In still yet another embodiments of the present aspect that can becombined with any of the preceding aspects, the antibody comprises an Fcregion. In certain embodiments, wherein the Fc region is a human Fcregion selected from the group consisting of an IgG1 Fc region, an IgG2Fc region, and an IgG4 Fc region. In some embodiments, the Fc region isa wild-type human IgG1, IgG2, or IgG4 Fc region. In some embodiments,the Fc region is a human Fc region comprising one or more amino acidsubstitutions that reduce one or more effector functions, as comparedwith the effector function(s) of a human Fc region that lacks the aminoacid substitution(s). In still further embodiments, the Fc region is:(a) a human IgG1 Fc region comprising L234A, L235A, and/or G237Asubstitutions, amino acid position numbering according to EU index; (b)a human IgG2 Fc region comprising A330S and/or P331S substitutions,amino acid position numbering according to EU index; or (c) a human IgG4Fc region comprising S228P and/or L235E substitutions, amino acidposition numbering according to EU index. In still yet anotherembodiment, the Fc region further comprises an N297A substitution, aminoacid position numbering according to EU index. In certain embodimentsthe Fc region further comprises an N297A substitution. In someembodiments, the antibody comprises an antibody heavy chain constantdomain comprising an amino acid sequence selected from the groupconsisting of SEQ ID Nos:92-107 and 178. In some embodiments, theconjugate further comprises an immunomodulating oligonucleotide Pattached to the Q295 residue of the Fc region as shown in the followingformula

wherein L is a linker moiety connected to Q295 via an amide bond. In yetother embodiments, the Fc region further comprises a D265A substitution,amino acid position numbering according to EU index. In someembodiments, the conjugate binds to human CD22 expressed on the surfaceof a B cell. In some embodiments, the conjugate induces activation ofTLR9.

In some embodiments of the present aspect, the antibody comprises ahuman lambda light chain. In other embodiments of the present aspect,the antibody comprises a human kappa light chain. In some embodiments,the antibody comprises an antibody light chain constant domaincomprising an amino acid sequence selected from the group consisting ofSEQ ID Nos:108-110. In still further embodiments which may be combinedwith any preceding embodiments of the present aspect, at least one Q-tagis attached to the heavy chain of the antibody. In certain embodiments,at least one Q-tag is fused to the C-terminus of the heavy chain of theantibody. In other embodiments, at least one Q-tag is attached to thelight chain of the antibody. In certain embodiments, the antibodycomprises two heavy chains and two light chains, and at least one Q-tagis fused to the C-terminus of each heavy chain.

In still further embodiments, at least one Q-tag is within the Fcdomain. In additional embodiments, each Q-tag independently comprises apeptide sequence having between 5 and 15 amino acid residues. In certainembodiments of the present aspect, the Q-tag is naturally occurring. Instill further embodiments, the peptide sequence of each Q-tag isindependently selected from the group consisting of SEQ ID NOs: 39-55.In certain embodiments, wherein the Q-tag comprises the peptide sequenceRPQGF (SEQ ID NO:47). In still yet other embodiments, 1 or 2 Q-tagsis/are linked to the antibody or antigen-binding fragment. In yet otherembodiments, the conjugate has a DAR of 1. In yet other embodiments, theconjugate has a DAR of 2. In additional embodiments which may becombined with any of the preceding embodiments, the linker L comprises apolyethylene glycol moiety. In yet further embodiments, the linker L is

wherein m is an integer ranging from about 0 to about 50, and wherein

indicates the point of attachment to Y^(PTE), and

indicates the point of attachment to the rest of the conjugate.

In some embodiments, Z is S. In still further embodiments, theoligonucleotide comprises at least one pair of geminal T¹ and T² whereinT¹ is S and T² is S⁻. In certain embodiments, the oligonucleotidecomprises at least two pairs of geminal T¹ and T² wherein T¹ is S and T²is S⁻. In still further embodiments, which may be combined with any ofthe preceding embodiments, R^(5′) is H. In other embodiments, R^(5′) ismethoxy. In some embodiments, R^(c1) is H. In yet other embodiments,R^(c1) is methoxy. In still further embodiments, R² is methyl. In stillother embodiments, R² is H. In additional embodiments, m is an integerfrom 20 to 25. In some embodiments of the present aspect, each Pindependently comprises an oligonucleotide sequence selected from thegroup consisting of the oligonucleotides of Table 10.

In some embodiments, the conjugate comprises one or more, two or more,three or more, four or more, five or more, or ten or more Q-tagpeptides. In some embodiments, the conjugate comprises two Q-tagpeptides. In some embodiments, the conjugate comprises one or more, twoor more, three or more, four or more, five or more, or ten or moreimmunomodulating oligonucleotides. In some embodiments, the conjugatecomprises one immunomodulating oligonucleotide. In other embodiments,the antibody is linked to 2 Q-tag peptides, and wherein one of the Q-tagpeptides is linked to an immunomodulating oligonucleotide. In someembodiments, the antibody comprises two antibody light chains, twoantibody heavy chains, and two Q-tag peptides; wherein each of the Q-tagpeptides is linked to the C-terminus of one of the antibody heavychains; and wherein at least one of the Q-tag peptides is linked to animmunomodulating oligonucleotide (P) via an amide bond with theglutamine residue of the Q-tag peptide and linker (L), e.g., as shown inFIGS. 16A-16D. In certain embodiments, the two Q-tag peptides comprisethe peptide sequence RPQGF (SEQ ID NO:47). In other embodiments, the twoQ-tag peptides comprise a Q295 residue exposed by N297A mutation (of theFc region). In still other embodiments, the conjugate has a DAR of 1 or2.

In yet another aspect, provided herein is a conjugate that comprises anantibody or antigen-binding fragment thereof (Ab) and one or moreimmunomodulating oligonucleotides (P), wherein the antibody orantigen-binding fragment is linked to one or more Q-tag peptides (Q)comprising the amino acid sequence RPQGF (SEQ ID NO:47), wherein eachimmunomodulating oligonucleotide is linked to a Q-tag peptide via anamide bond with the glutamine residue of the Q-tag peptide and a linker(L) as shown in formula (A),

wherein:

indicates the point of attachment of each Q to the antibody orantigen-binding fragment thereof (Ab);each Q comprises a Q-tag peptide sequence RPQGF (SEQ ID NO:47);each L is independently a bond or a linker moiety

wherein m is an integer ranging from about 0 to about 50, and wherein

indicates the point of attachment to P, and

indicates the point of attachment to the rest of the conjugate connectedto Q via an amide bond with glutamine residue;and each P is an immunomodulating oligonucleotide having the structure

wherein

and

indicate the points of attachment within the oligonucleotide; wherein Abcomprises a heavy chain variable (VH) domain and a light chain variable(VL) domain, wherein the VH domain comprises CDR-H1, CDR-H2, and CDR-H3sequences from a VH domain sequenceQVQLLESGGGVVQPGGSLRLSCAASGFAFSIYDMNWVRQAPGKGLEWVSAISSGGGTTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHSGYGTHWGVLFAYWG RGTLVTVSS (SEQID NO: 65) or a VH domain shown in Table 8;wherein the VL domain comprises CDR-L1, CDR-L2, and CDR-L3 sequencesfrom a VL domain sequence:DIQMTQSPSSLSASVGDRVTITCRASQDIHGYLNWYQQKPGKAPKLLIYYTSILHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQGNTLPWTFGQGTKLEIK (SEQ ID NO:68) or a VLdomain shown in Table 8.

In some embodiments of the present aspect, the VL domain furthercomprises an amino acid substitution N92A. In other embodiments, the VLdomain further comprises an amino acid substitution N92L. In still otherembodiments, the VL domain further comprises an amino acid substitutionN92S.

In other embodiments of the present aspect, each Q tag is independentlyselected from the group consisting of RPQGF (SEQ ID NO:47), RPQGFPP (SEQID NO:48), and RPQGFGPP (SEQ ID NO:49). In certain embodiments, each Qtag is RPQGFGPP (SEQ ID NO:49). In some embodiments, 1 or 2 Q-tags arelinked to the antibody or antigen-binding fragment. In otherembodiments, the Q-tag is linked to the C-terminus of the heavy chain ofthe antibody. In still further embodiments, the antibody comprises ahuman IgG1 Fc region comprising L234A, L235A, and/or G237Asubstitutions, amino acid position numbering according to EU index. Inyet other embodiments, m is an integer from about 20 to about 25. Incertain embodiments, m is 24. In yet further embodiments, the conjugatehas a DAR of 1. In yet further embodiments, the conjugate has a DAR of2. In still other embodiments, the conjugate binds to human CD22expressed on the surface of a B cell.

In another aspect, provided herein is a conjugate that comprises anantibody or antigen-binding fragment thereof (Ab) and one or moreimmunomodulating oligonucleotides (P), wherein the antibody orantigen-binding fragment is linked to one or more Q-tag peptides (Q)comprising the amino acid sequence RPQGF (SEQ ID NO:47), wherein eachimmunomodulating oligonucleotide is linked to a Q-tag peptide via anamide bond with the glutamine residue of the Q-tag peptide and a linker(L) as shown in formula (A),

wherein:

indicates the point of attachment of each Q to the antibody orantigen-binding fragment thereof (Ab);each Q comprises a Q-tag peptide sequence RPQGF (SEQ ID NO:47);each L is independently a bond or a linker moiety

wherein m is an integer ranging from about 0 to about 50, and wherein

indicates the point of attachment to P, and

indicates the point of attachment to the rest of the conjugate connectedto Q via an amide bond with the glutamine residue;and each P is an immunomodulating oligonucleotide having the structure

wherein

and

indicate the points of attachment within the oligonucleotide;wherein Ab comprises a heavy chain variable (VH) domain and a lightchain variable (VL) domain, wherein the VH domain comprises CDR-H1,CDR-H2, and CDR-H3 sequences from a VH domain sequence

(SEQ ID NO: 65) QVQLLESGGGVVQPGGSLRLSCAASGFAFSIYDMNWVRQAPGKGLEWVSAISSGGGTTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHSGYGTHWGVLFAYWGRGTLVTVSS;wherein the VL domain comprises CDR-L1, CDR-L2, and CDR-L3 sequencesfrom a VL domain sequence:

(SEQ ID NO: 68) DIQMTQSPSSLSASVGDRVTITCRASQDIHGYLNWYQQKPGKAPKLLIYYTSILHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQGNTLPWTFGQ GTKLEIK.

In some embodiments of the present aspect, the VL domain furthercomprises an amino acid substitution N92A. In other embodiments, the VLdomain further comprises an amino acid substitution N92L. In still otherembodiments, the VL domain further comprises an amino acid substitutionN92S.

In other embodiments of the present aspect, each Q tag is independentlyselected from the group consisting of RPQGF (SEQ ID NO:47), RPQGFPP (SEQID NO:48), and RPQGFGPP. In certain embodiments, each Q tag is RPQGFGPP(SEQ ID NO:49). In some embodiments, 1 or 2 Q-tags are linked to theantibody or antigen-binding fragment. In other embodiments, the Q-tag islinked to the C-terminus of the heavy chain of the antibody. In stillfurther embodiments, the antibody comprises a human IgG1 Fc regioncomprising L234A, L235A, and/or G237A substitutions, amino acid positionnumbering according to EU index. In yet other embodiments, m is aninteger from about 20 to about 25. In certain embodiments, m is 24. Inyet further embodiments, the conjugate has a DAR of 1. In yet furtherembodiments, the conjugate has a DAR of 2. In still other embodiments,the conjugate binds to human CD22 expressed on the surface of a B cell.

In still yet another aspect, the present disclosure also provides animmunomodulating oligonucleotide of Formula (C):

wherein

and

indicate the points of attachment within the oligonucleotide;

each T¹ is independently O or S;

each T² is S⁻;

T³ is a group

wherein m is an integer from 0 to 50 and wherein

indicates the point of attachment to the rest of the oligonucleotide;

Z is O or S;

U^(5′) is —H or halogen;

R^(5′) is —H or methoxy;

R^(c1) is —H or methoxy;

R^(g1), R^(g2), R^(g3), and R^(g4) are H;

R^(3′) is methoxy;

R¹ is —(CH₂)₃—OH;

R² is —H or methyl; and

n is an integer from 0 to 2,

or a pharmaceutically acceptable salt thereof.

In some embodiments, the present disclosure also provides animmunomodulating oligonucleotide of Formula (C′):

wherein

and

indicate the points of attachment within the oligonucleotide;

each T¹ is independently O or S;

each T² is S⁻;

T³ is a group

wherein m is an integer from 0 to 50 and wherein

indicates the point of attachment to the rest of the oligonucleotide;

Z is O or S;

R^(5′) is —H or methoxy;

R^(c1) is —H or methoxy;

R^(g1), R^(g2), R^(g3), and R^(g4) are H;

R^(3′) is methoxy;

R¹ is —(CH₂)₃—OH;

R² is —H or methyl; and

n is an integer from 0 to 2.

In some embodiments of the present aspect, Z is S. In additionalembodiments, the oligonucleotide comprises at least one pair of geminalT¹ and T² wherein T¹ is S and T² is S. In certain embodiments, theoligonucleotide comprises at least two pairs of geminal T¹ and T²wherein T¹ is S and T² is S⁻.

In some embodiments, R^(5′) is H. In other embodiments, R^(5′) ismethoxy. In some embodiments, R^(c1) is H. In yet other embodiments,R^(c1) is methoxy. In still further embodiments, R² is methyl. In stillother embodiments, R² is H. In yet other additional embodiments, whichmay be combined with any of the preceding embodiments, T³ is

In still other embodiments, T³ is

In certain embodiments, m is an integer from 20 to 25.

In still further embodiments of the present aspect, the oligonucleotideis selected from the group consisting of the oligonucleotides of Table10.

In still yet another aspect, provided herein is an immunomodulatingoligonucleotide of formula (D):

wherein

and

indicate the points of attachment within the oligonucleotide;

each T¹ is independently O or S;

each T² is S⁻;

T³ is a group

wherein

indicates the point of attachment to L and wherein

indicates the point of attachment to the rest of the oligonucleotide;

L is a group

wherein m is an integer from 0 to 50 and wherein

indicates the point of attachment to the rest of the oligonucleotide viaT³;

Z is O or S;

U^(5′) is —H or halogen;

R^(5′) is —H or methoxy;

R is —H or methoxy;

R^(g1), R^(g2), R^(g3), and R^(g4) are H;

R^(3′) is methoxy;

R¹ is —(CH₂)₃—OH;

R² is —H or methyl; and

n is an integer from 0 to 2.

In some embodiments, provided herein is an immunomodulatingoligonucleotide of formula (D′):

wherein

and

indicate the points of attachment within the oligonucleotide;

each T¹ is independently O or S;

each T² is S⁻;

T³ is a group

wherein

indicates the point of attachment to L and wherein

indicates the point of attachment to the rest of the oligonucleotide;

L is a group

wherein m is an integer from 0 to 50 and wherein

indicates the point of attachment to the rest of the oligonucleotide viaT³

Z is O or S;

R^(5′) is —H or methoxy;

R^(c1) is —H or methoxy;

R^(g1), R^(g2), R^(g3), and R^(g4) are H;

R^(3′) is methoxy;

R¹ is —(CH₂)₃—OH;

R² is —H or methyl; and

n is an integer from 0 to 2.

In some embodiments of the present aspect, Z is S. In additionalembodiments, the oligonucleotide comprises at least one pair of geminalT¹ and T² wherein T¹ is S and T² is S⁻. In certain embodiments, theoligonucleotide comprises at least two pairs of geminal T¹ and T²wherein T¹ is S and T² is S⁻.

In some embodiments, R^(5′) is H. In other embodiments, R^(5′) ismethoxy. In some embodiments, R^(c1) is H. In yet other embodiments,R^(c1) is methoxy. In still further embodiments, R² is methyl. In stillother embodiments, R² is H. In certain embodiments, m is an integer from20 to 25.

In still further embodiments of the present aspect, the oligonucleotideis selected from the group consisting of the oligonucleotides of Table12.

In yet another aspect, provided herein is an immunomodulatingoligonucleotide selected from the group consisting of theoligonucleotides of Table 10 and Table 12.

In still yet another aspect, the present disclosure provides a conjugatecomprising a protein, at least one Q tag peptide sequence comprising aglutamine residue, and at least one immunomodulatory oligonucleotide,wherein the Q-tag peptide sequence is naturally occurring or synthetic,and wherein the immunomodulatory oligonucleotide is linked to the Q-tagvia an amide bond with the glutamine residue, wherein the Q-tag peptidesequence is selected from the group consisting of SEQ ID NOs: 39-55. Insome embodiments of the present aspect, immunomodulatory oligonucleotidehas a sequence selected from the group consisting of theoligonucleotides of Table 10 and Table 12. In further embodiments, theantibody comprises a light chain variable domain (VL) and a heavy chainvariable domain (VH), and wherein VH comprises the sequence SEQ ID NO:56; and VL comprises the sequence SEQ ID NO: 57.

In still yet another aspect, the present disclosure provides a conjugatecomprising an antibody linked to two Q-tag peptides (Q) and animmunomodulating oligonucleotide (P); wherein the antibody comprises twoantibody light chains, two antibody heavy chains, and two Q-tagpeptides; wherein each of the Q-tag peptides is linked to the C-terminusof one of the antibody heavy chains; and wherein one of the Q-tagpeptides is linked to the immunomodulating oligonucleotide via an amidebond with the glutamine residue of the Q-tag peptide and a linker (L) asshown in Formula (A):

wherein

indicates the point of attachment of each Q to the antibody.

In some embodiments, each Q-tag peptide comprises the amino acidsequence RPQGF (SEQ ID NO:47). In some embodiments, each L is a linkeras described herein. In some embodiments, each (P) is animmunomodulating oligonucleotide as described herein. In someembodiments, the antibody is an antibody as described herein. In someembodiments, the antibody binds to CD22 (e.g., human CD22).

In yet another aspect, provided herein is a conjugate comprising anantibody (Ab) and an immunomodulating oligonucleotide (P), wherein theantibody comprises two antibody light chains, two antibody heavy chains,and two Q-tag peptides; wherein each of the Q-tag peptides (Q) comprisesthe amino acid sequence RPQGF (SEQ ID NO:47); wherein each of the Q-tagpeptides is linked to the C-terminus of one of the antibody heavychains; wherein one of the two Q-tag peptides is linked to theimmunomodulating oligonucleotide via an amide bond with the glutamineresidue of the Q-tag peptide and a linker (L) as shown in Formula (A):

wherein

indicates the point of attachment of each Q to the antibody (Ab).

In yet another aspect, provided herein is a conjugate comprising ananti-CD22 antibody (Ab) and an immunomodulating oligonucleotide (P),wherein the antibody comprises two antibody light chains, two antibodyheavy chains, and two Q-tag peptides; wherein each of the Q-tag peptides(Q) comprises the amino acid sequence of SEQ ID NO:49; wherein each ofthe Q-tag peptides is linked to the C-terminus of one of the antibodyheavy chains; wherein one of the two Q-tag peptides is linked to theimmunomodulating oligonucleotide via an amide bond with the glutamineresidue of the Q-tag peptide and a linker (L), wherein the two antibodyheavy chains comprise a VH domain comprising the sequence of SEQ IDNO:65 and a constant region comprising the sequence of SEQ ID NO:92,wherein the two antibody light chains comprise a VL domain comprisingthe sequence of SEQ ID NO:87 and a constant region comprising thesequence of SEQ ID NO:108, and wherein the immunomodulatingoligonucleotide comprises the sequence of SEQ ID NO:35. In yet anotheraspect, provided herein is a conjugate comprising an anti-CD22 antibody(Ab) and an immunomodulating oligonucleotide (P), wherein the antibodycomprises two antibody light chains, two antibody heavy chains, and twoQ-tag peptides; wherein each of the Q-tag peptides (Q) comprises theamino acid sequence of SEQ ID NO:49; wherein each of the Q-tag peptidesis linked to the C-terminus of one of the antibody heavy chains; whereinone of the two Q-tag peptides is linked to the immunomodulatingoligonucleotide via an amide bond with the glutamine residue of theQ-tag peptide and a linker (L), wherein the two antibody heavy chainscomprise a VH domain comprising the sequence of SEQ ID NO:65 and aconstant region comprising the sequence of SEQ ID NO:94, wherein the twoantibody light chains comprise a VL domain comprising the sequence ofSEQ ID NO:87 and a constant region comprising the sequence of SEQ IDNO:108, and wherein the immunomodulating oligonucleotide comprises thesequence of SEQ ID NO:35. In yet another aspect, provided herein is aconjugate comprising an anti-CD22 antibody (Ab) and an immunomodulatingoligonucleotide (P), wherein the antibody comprises two antibody lightchains, two antibody heavy chains, and two Q-tag peptides; wherein eachof the Q-tag peptides (Q) comprises the amino acid sequence of SEQ IDNO:49; wherein each of the Q-tag peptides is linked to the C-terminus ofone of the antibody heavy chains; wherein one of the two Q-tag peptidesis linked to the immunomodulating oligonucleotide via an amide bond withthe glutamine residue of the Q-tag peptide and a linker (L), wherein thetwo antibody heavy chains comprise a VH domain comprising the sequenceof SEQ ID NO:65 and a constant region comprising the sequence of SEQ IDNO:94, wherein the two antibody light chains comprise a VL domaincomprising the sequence of SEQ ID NO:73 and a constant region comprisingthe sequence of SEQ ID NO:108, and wherein the immunomodulatingoligonucleotide comprises the sequence of SEQ ID NO:34. In yet anotheraspect, provided herein is a conjugate comprising an anti-CD22 antibody(Ab) and an immunomodulating oligonucleotide (P), wherein the antibodycomprises two antibody light chains, two antibody heavy chains, and twoQ-tag peptides; wherein each of the Q-tag peptides (Q) comprises theamino acid sequence of SEQ ID NO:49; wherein each of the Q-tag peptidesis linked to the C-terminus of one of the antibody heavy chains; whereinone of the two Q-tag peptides is linked to the immunomodulatingoligonucleotide via an amide bond with the glutamine residue of theQ-tag peptide and a linker (L), wherein the two antibody heavy chainscomprise a VH domain comprising the sequence of SEQ ID NO:65 and aconstant region comprising the sequence of SEQ ID NO:92, wherein the twoantibody light chains comprise a VL domain comprising the sequence ofSEQ ID NO:73 and a constant region comprising the sequence of SEQ IDNO:108, and wherein the immunomodulating oligonucleotide comprises thesequence of SEQ ID NO:34. In yet another aspect, provided herein is aconjugate comprising an anti-CD22 antibody (Ab) and an immunomodulatingoligonucleotide (P), wherein the antibody comprises two antibody lightchains, two antibody heavy chains, and two Q-tag peptides; wherein eachof the Q-tag peptides (Q) comprises the amino acid sequence of SEQ IDNO:49; wherein each of the Q-tag peptides is linked to the C-terminus ofone of the antibody heavy chains; wherein one of the two Q-tag peptidesis linked to the immunomodulating oligonucleotide via an amide bond withthe glutamine residue of the Q-tag peptide and a linker (L), wherein thetwo antibody heavy chains comprise a VH domain comprising the sequenceof SEQ ID NO:65 and a constant region comprising the sequence of SEQ IDNO:92, wherein the two antibody light chains comprise a VL domaincomprising the sequence of SEQ ID NO:73 and a constant region comprisingthe sequence of SEQ ID NO:108, and wherein the immunomodulatingoligonucleotide comprises the sequence of SEQ ID NO:163. In yet anotheraspect, provided herein is a conjugate comprising an anti-CD22 antibody(Ab) and an immunomodulating oligonucleotide (P), wherein the antibodycomprises two antibody light chains, two antibody heavy chains, and twoQ-tag peptides; wherein each of the Q-tag peptides (Q) comprises theamino acid sequence of SEQ ID NO:49; wherein each of the Q-tag peptidesis linked to the C-terminus of one of the antibody heavy chains; whereinone of the two Q-tag peptides is linked to the immunomodulatingoligonucleotide via an amide bond with the glutamine residue of theQ-tag peptide and a linker (L), wherein the two antibody heavy chainscomprise a VH domain comprising the sequence of SEQ ID NO:65 and aconstant region comprising the sequence of SEQ ID NO:92, wherein the twoantibody light chains comprise a VL domain comprising the sequence ofSEQ ID NO:87 and a constant region comprising the sequence of SEQ IDNO:108, and wherein the immunomodulating oligonucleotide comprises thesequence of SEQ ID NO:163. In some embodiments, the linker comprises thelinker moiety

wherein m is an integer 24, and wherein

indicates the point of attachment to P, and

indicates the point of attachment to the rest of the conjugate connectedto Q via an amide bond with the glutamine residue.

In yet another aspect, provided herein is a conjugate comprising ananti-Her2 antibody (Ab) and an immunomodulating oligonucleotide (P),wherein the antibody comprises two antibody light chains, two antibodyheavy chains, and two Q-tag peptides; wherein each of the Q-tag peptides(Q) comprises the amino acid sequence of SEQ ID NO:49; wherein each ofthe Q-tag peptides is linked to the C-terminus of one of the antibodyheavy chains; wherein one of the two Q-tag peptides is linked to theimmunomodulating oligonucleotide via an amide bond with the glutamineresidue of the Q-tag peptide and a linker (L), wherein the two antibodyheavy chains comprise a VH domain comprising the sequence of SEQ IDNO:168 and a constant region comprising the sequence of SEQ ID NO:92,wherein the two antibody light chains comprise a VL domain comprisingthe sequence of SEQ ID NO:169 and a constant region comprising thesequence of SEQ ID NO:108, and wherein the immunomodulatingoligonucleotide comprises the sequence of SEQ ID NO:35. In yet anotheraspect, provided herein is a conjugate comprising an anti-Her2 antibody(Ab) and an immunomodulating oligonucleotide (P), wherein the antibodycomprises two antibody light chains, two antibody heavy chains, and twoQ-tag peptides; wherein each of the Q-tag peptides (Q) comprises theamino acid sequence of SEQ ID NO:49; wherein each of the Q-tag peptidesis linked to the C-terminus of one of the antibody heavy chains; whereinone of the two Q-tag peptides is linked to the immunomodulatingoligonucleotide via an amide bond with the glutamine residue of theQ-tag peptide and a linker (L), wherein the two antibody heavy chainscomprise a VH domain comprising the sequence of SEQ ID NO:168 and aconstant region comprising the sequence of SEQ ID NO:104, wherein thetwo antibody light chains comprise a VL domain comprising the sequenceof SEQ ID NO:169 and a constant region comprising the sequence of SEQ IDNO:108, and wherein the immunomodulating oligonucleotide comprises thesequence of SEQ ID NO:35. In yet another aspect, provided herein is aconjugate comprising an anti-Her2 antibody (Ab) and an immunomodulatingoligonucleotide (P), wherein the antibody comprises two antibody lightchains, two antibody heavy chains, and two Q-tag peptides; wherein eachof the Q-tag peptides (Q) comprises the amino acid sequence of SEQ IDNO:49; wherein each of the Q-tag peptides is linked to the C-terminus ofone of the antibody heavy chains; wherein one of the two Q-tag peptidesis linked to the immunomodulating oligonucleotide via an amide bond withthe glutamine residue of the Q-tag peptide and a linker (L), wherein thetwo antibody heavy chains comprise a VH domain comprising the sequenceof SEQ ID NO:168 and a constant region comprising the sequence of SEQ IDNO:92, wherein the two antibody light chains comprise a VL domaincomprising the sequence of SEQ ID NO:169 and a constant regioncomprising the sequence of SEQ ID NO:108, and wherein theimmunomodulating oligonucleotide comprises the sequence of SEQ IDNO:163. In yet another aspect, provided herein is a conjugate comprisingan anti-Her2 antibody (Ab) and an immunomodulating oligonucleotide (P),wherein the antibody comprises two antibody light chains, two antibodyheavy chains, and two Q-tag peptides; wherein each of the Q-tag peptides(Q) comprises the amino acid sequence of SEQ ID NO:49; wherein each ofthe Q-tag peptides is linked to the C-terminus of one of the antibodyheavy chains; wherein one of the two Q-tag peptides is linked to theimmunomodulating oligonucleotide via an amide bond with the glutamineresidue of the Q-tag peptide and a linker (L), wherein the two antibodyheavy chains comprise a VH domain comprising the sequence of SEQ IDNO:170 and a constant region comprising the sequence of SEQ ID NO:92,wherein the two antibody light chains comprise a VL domain comprisingthe sequence of SEQ ID NO:171 and a constant region comprising thesequence of SEQ ID NO:108, and wherein the immunomodulatingoligonucleotide comprises the sequence of SEQ ID NO:35. In yet anotheraspect, provided herein is a conjugate comprising an anti-Her2 antibody(Ab) and an immunomodulating oligonucleotide (P), wherein the antibodycomprises two antibody light chains, two antibody heavy chains, and twoQ-tag peptides; wherein each of the Q-tag peptides (Q) comprises theamino acid sequence of SEQ ID NO:49; wherein each of the Q-tag peptidesis linked to the C-terminus of one of the antibody heavy chains; whereinone of the two Q-tag peptides is linked to the immunomodulatingoligonucleotide via an amide bond with the glutamine residue of theQ-tag peptide and a linker (L), wherein the two antibody heavy chainscomprise a VH domain comprising the sequence of SEQ ID NO:170 and aconstant region comprising the sequence of SEQ ID NO:104, wherein thetwo antibody light chains comprise a VL domain comprising the sequenceof SEQ ID NO:171 and a constant region comprising the sequence of SEQ IDNO:108, and wherein the immunomodulating oligonucleotide comprises thesequence of SEQ ID NO:35. In yet another aspect, provided herein is aconjugate comprising an anti-Her2 antibody (Ab) and an immunomodulatingoligonucleotide (P), wherein the antibody comprises two antibody lightchains, two antibody heavy chains, and two Q-tag peptides; wherein eachof the Q-tag peptides (Q) comprises the amino acid sequence of SEQ IDNO:49; wherein each of the Q-tag peptides is linked to the C-terminus ofone of the antibody heavy chains; wherein one of the two Q-tag peptidesis linked to the immunomodulating oligonucleotide via an amide bond withthe glutamine residue of the Q-tag peptide and a linker (L), wherein thetwo antibody heavy chains comprise a VH domain comprising the sequenceof SEQ ID NO:170 and a constant region comprising the sequence of SEQ IDNO:92, wherein the two antibody light chains comprise a VL domaincomprising the sequence of SEQ ID NO:171 and a constant regioncomprising the sequence of SEQ ID NO:108, and wherein theimmunomodulating oligonucleotide comprises the sequence of SEQ IDNO:163. In some embodiments, the linker comprises the linker moiety

wherein m is an integer 24, and wherein

indicates the point of attachment to P, and

indicates the point of attachment to the rest of the conjugate connectedto Q via an amide bond with the glutamine residue.

In yet another aspect, provided herein is a conjugate comprising ananti-CD22 antibody (Ab) and an immunomodulating oligonucleotide (P),wherein the antibody comprises two antibody light chains, two antibodyheavy chains, and two Q-tag peptides; wherein each of the antibody lightchains comprises the amino acid sequence of SEQ ID NO:182; wherein eachof the antibody heavy chains comprises the amino acid sequence of SEQ IDNO:179; wherein the immunomodulating oligonucleotide comprises thesequence of SEQ ID NO:35; and wherein at least one of the two Q-tagpeptides is linked to the immunomodulating oligonucleotide via an amidebond with the glutamine residue of the Q-tag peptide and a linker (L).In yet another aspect, provided herein is a conjugate comprising anantibody (Ab) and an immunomodulating oligonucleotide (P), wherein theantibody comprises two antibody light chains, two antibody heavy chains,and two Q-tag peptides; wherein each of the antibody light chainscomprises the amino acid sequence of SEQ ID NO:182; wherein each of theantibody heavy chains comprises the amino acid sequence of SEQ IDNO:180; wherein the immunomodulating oligonucleotide comprises thesequence of SEQ ID NO:35; and wherein at least one of the two Q-tagpeptides is linked to the immunomodulating oligonucleotide via an amidebond with the glutamine residue of the Q-tag peptide and a linker (L).In yet another aspect, provided herein is a conjugate comprising ananti-CD22 antibody (Ab) and an immunomodulating oligonucleotide (P),wherein the antibody comprises two antibody light chains, two antibodyheavy chains, and two Q-tag peptides; wherein each of the antibody lightchains comprises the amino acid sequence of SEQ ID NO:181; wherein eachof the antibody heavy chains comprises the amino acid sequence of SEQ IDNO: 180; wherein the immunomodulating oligonucleotide comprises thesequence of SEQ ID NO:34; and wherein at least one of the two Q-tagpeptides is linked to the immunomodulating oligonucleotide via an amidebond with the glutamine residue of the Q-tag peptide and a linker (L).In yet another aspect, provided herein is a conjugate comprising ananti-CD22 antibody (Ab) and an immunomodulating oligonucleotide (P),wherein the antibody comprises two antibody light chains, two antibodyheavy chains, and two Q-tag peptides; wherein each of the antibody lightchains comprises the amino acid sequence of SEQ ID NO:181; wherein eachof the antibody heavy chains comprises the amino acid sequence of SEQ IDNO:179; wherein the immunomodulating oligonucleotide comprises thesequence of SEQ ID NO:34; and wherein at least one of the two Q-tagpeptides is linked to the immunomodulating oligonucleotide via an amidebond with the glutamine residue of the Q-tag peptide and a linker (L).In yet another aspect, provided herein is a conjugate comprising ananti-CD22 antibody (Ab) and an immunomodulating oligonucleotide (P),wherein the antibody comprises two antibody light chains, two antibodyheavy chains, and two Q-tag peptides; wherein each of the antibody lightchains comprises the amino acid sequence of SEQ ID NO:181; wherein eachof the antibody heavy chains comprises the amino acid sequence of SEQ IDNO: 179; wherein the immunomodulating oligonucleotide comprises thesequence of SEQ ID NO:163; and wherein at least one of the two Q-tagpeptides is linked to the immunomodulating oligonucleotide via an amidebond with the glutamine residue of the Q-tag peptide and a linker (L).In yet another aspect, provided herein is a conjugate comprising ananti-CD22 antibody (Ab) and an immunomodulating oligonucleotide (P),wherein the antibody comprises two antibody light chains, two antibodyheavy chains, and two Q-tag peptides; wherein each of the antibody lightchains comprises the amino acid sequence of SEQ ID NO:182; wherein eachof the antibody heavy chains comprises the amino acid sequence of SEQ IDNO:179; wherein the immunomodulating oligonucleotide comprises thesequence of SEQ ID NO:163; and wherein at least one of the two Q-tagpeptides is linked to the immunomodulating oligonucleotide via an amidebond with the glutamine residue of the Q-tag peptide and a linker (L).In some embodiments, one of the two Q-tag peptides is linked to theimmunomodulating oligonucleotide. In some embodiments, the conjugatecomprises two immunomodulating oligonucleotides, wherein each of the twoQ-tag peptides is linked to one of the two immunomodulatingoligonucleotides. In some embodiments, the conjugate has a DAR of 1. Insome embodiments, the conjugate has a DAR of 2. In some embodiments, thelinker comprises the linker moiety

wherein m is an integer 24, and wherein

indicates the point of attachment to P, and

indicates the point of attachment to the rest of the conjugate connectedto Q via an amide bond with the glutamine residue.

In yet another aspect, provided herein is a conjugate comprising ananti-Her2 antibody (Ab) and an immunomodulating oligonucleotide (P),wherein the antibody comprises two antibody light chains, two antibodyheavy chains, and two Q-tag peptides; wherein each of the antibody lightchains comprises the amino acid sequence of SEQ ID NO:185; wherein eachof the antibody heavy chains comprises the amino acid sequence of SEQ IDNO:184; wherein the immunomodulating oligonucleotide comprises thesequence of SEQ ID NO:35; and wherein at least one of the two Q-tagpeptides is linked to the immunomodulating oligonucleotide via an amidebond with the glutamine residue of the Q-tag peptide and a linker (L).In yet another aspect, provided herein is a conjugate comprising ananti-Her2 antibody (Ab) and an immunomodulating oligonucleotide (P),wherein the antibody comprises two antibody light chains, two antibodyheavy chains, and two Q-tag peptides; wherein each of the antibody lightchains comprises the amino acid sequence of SEQ ID NO:185; wherein eachof the antibody heavy chains comprises the amino acid sequence of SEQ IDNO:183; wherein the immunomodulating oligonucleotide comprises thesequence of SEQ ID NO:35; and wherein at least one of the two Q-tagpeptides is linked to the immunomodulating oligonucleotide via an amidebond with the glutamine residue of the Q-tag peptide and a linker (L).In yet another aspect, provided herein is a conjugate comprising ananti-Her2 antibody (Ab) and an immunomodulating oligonucleotide (P),wherein the antibody comprises two antibody light chains, two antibodyheavy chains, and two Q-tag peptides; wherein each of the antibody lightchains comprises the amino acid sequence of SEQ ID NO:185; wherein eachof the antibody heavy chains comprises the amino acid sequence of SEQ IDNO: 184; wherein the immunomodulating oligonucleotide comprises thesequence of SEQ ID NO: 163; and wherein at least one of the two Q-tagpeptides is linked to the immunomodulating oligonucleotide via an amidebond with the glutamine residue of the Q-tag peptide and a linker (L).In yet another aspect, provided herein is a conjugate comprising ananti-Her2 antibody (Ab) and an immunomodulating oligonucleotide (P),wherein the antibody comprises two antibody light chains, two antibodyheavy chains, and two Q-tag peptides; wherein each of the antibody lightchains comprises the amino acid sequence of SEQ ID NO:188; wherein eachof the antibody heavy chains comprises the amino acid sequence of SEQ IDNO:187; wherein the immunomodulating oligonucleotide comprises thesequence of SEQ ID NO:35; and wherein at least one of the two Q-tagpeptides is linked to the immunomodulating oligonucleotide via an amidebond with the glutamine residue of the Q-tag peptide and a linker (L).In yet another aspect, provided herein is a conjugate comprising ananti-Her2 antibody (Ab) and an immunomodulating oligonucleotide (P),wherein the antibody comprises two antibody light chains, two antibodyheavy chains, and two Q-tag peptides; wherein each of the antibody lightchains comprises the amino acid sequence of SEQ ID NO:188; wherein eachof the antibody heavy chains comprises the amino acid sequence of SEQ IDNO: 186; wherein the immunomodulating oligonucleotide comprises thesequence of SEQ ID NO:35; and wherein at least one of the two Q-tagpeptides is linked to the immunomodulating oligonucleotide via an amidebond with the glutamine residue of the Q-tag peptide and a linker (L).In yet another aspect, provided herein is a conjugate comprising ananti-Her2 antibody (Ab) and an immunomodulating oligonucleotide (P),wherein the antibody comprises two antibody light chains, two antibodyheavy chains, and two Q-tag peptides; wherein each of the antibody lightchains comprises the amino acid sequence of SEQ ID NO:188; wherein eachof the antibody heavy chains comprises the amino acid sequence of SEQ IDNO:187; wherein the immunomodulating oligonucleotide comprises thesequence of SEQ ID NO:163; and wherein at least one of the two Q-tagpeptides is linked to the immunomodulating oligonucleotide via an amidebond with the glutamine residue of the Q-tag peptide and a linker (L).In some embodiments, one of the two Q-tag peptides is linked to theimmunomodulating oligonucleotide. In some embodiments, the conjugatecomprises two immunomodulating oligonucleotides, wherein each of the twoQ-tag peptides is linked to one of the two immunomodulatingoligonucleotides. In some embodiments, the conjugate has a DAR of 1. Insome embodiments, the conjugate has a DAR of 2. In some embodiments, thelinker comprises the linker moiety

wherein m is an integer 24, and wherein

indicates the point of attachment to P, and

indicates the point of attachment to the rest of the conjugate connectedto Q via an amide bond with the glutamine residue.

In yet another aspect, provided herein is a conjugate that comprises anantibody (Ab) and one or more immunomodulating oligonucleotides (P),wherein the antibody is linked to one or more Q-tag peptides (Q)comprising at least one glutamine residue, wherein each immunomodulatingoligonucleotide is linked to a Q-tag peptide via an amide bond with theglutamine residue of the Q-tag peptide and a linker (L) as shown informula (A),

wherein:wherein

indicates the point of attachment of Q to the antibody (Ab);the antibody comprise two antibody light chains, two antibody heavychains, and two Q-tag peptides;each of the Q-tag peptides is linked to the C-terminus of one of theantibody heavy chains; one of the two Q-tag peptides is linked to theimmunomodulating oligonucleotide via an amide bond with the glutamineresidue of the Q-tag peptide;each Q is independently a Q-tag peptide sequence having at least oneglutamine residue;each L is independently a bond or a linker moiety connected to Q via anamide bond with the glutamine residue; andeach P is independently an immunomodulating oligonucleotide having thestructure

wherein

and

indicates the points of attachment within the oligonucleotide;

each T¹ is independently O or S;each T² is S⁻;T³ is a group

wherein

indicates the point of attachment to L and wherein

indicates the point of attachment to the rest of the oligonucleotide;Z is O or S;

U^(5′) is —H or halogen;R^(5′) is —H or methoxy;R^(c1) is —H or methoxy;R^(g1), R^(g2), R^(g3), and R^(g4) are H;R^(3′) is methoxy;R¹ is —(CH₂)₃—OH;R² is —H or methyl; andn is an integer from 0 to 2.

In yet another aspect, provided herein is a conjugate that comprises anantibody (Ab) and one or more immunomodulating oligonucleotides (P),wherein the antibody is linked to one or more Q-tag peptides (Q)comprising at least one glutamine residue, wherein each immunomodulatingoligonucleotide is linked to a Q-tag peptide via an amide bond with theglutamine residue of the Q-tag peptide and a linker (L) as shown informula (A),

wherein:wherein

indicates the point of attachment of Q to the antibody (Ab);the antibody comprises two antibody light chains, two antibody heavychains, and two Q-tag peptides;each of the Q-tag peptides is linked to the C-terminus of one of theantibody heavy chains; one of the two Q-tag peptides is linked to theimmunomodulating oligonucleotide via an amide bond with the glutamineresidue of the Q-tag peptide;each Q is independently a Q-tag peptide sequence having at least oneglutamine residue;each L is independently a bond or a linker moiety connected to Q via anamide bond with the glutamine residue; andeach P is independently an immunomodulating oligonucleotide having thestructure

wherein

and

indicate the points of attachment within the oligonucleotide;

each T¹ is independently O or S;each T² is S⁻;T³ is a group

wherein

indicates the point of attachment to L and wherein

indicates the point of attachment to the rest of the oligonucleotide;

Z is O or S;

R^(5′) is —H or methoxy;R^(c1) is —H or methoxy;R^(g1), R^(g2), R^(g3), and R^(g4) are H;R^(3′) is methoxy;R¹ is —(CH₂)₃—OH;R² is —H or methyl; andn is an integer from 0 to 2.

In still another aspect, provided herein is a conjugate comprising anantibody or antigen-binding fragment thereof (Ab) and one or moreimmunomodulating oligonucleotides (P), wherein the antibody orantigen-binding fragment is linked to one or more Q-tag peptides (Q)comprising a Q-tag peptide sequence RPQGF (SEQ ID NO:47), and whereineach immunomodulating oligonucleotide is linked to a Q-tag peptide viaan amide bond with the glutamine residue of the Q-tag peptide and alinker (L) as shown in Formula (A)

wherein:

indicates the point of attachment of each Q to the antibody orantigen-binding fragment thereof (Ab)

each Q independently comprises a Q-tag peptide sequence RPQGF (SEQ IDNO:47);

each L is independently a bond or a linker moiety

wherein m is an integer ranging from about 0 to about 50, and wherein

indicates the point of attachment to P, and

indicates the point of attachment to the rest of the conjugate connectedto Q via an amide bond with the glutamine residue; and

each P is independently an immunomodulating oligonucleotide having thestructure

wherein

and

indicate the points of attachment within the oligonucleotide;

each T¹ is independently O or S;

each T² is S⁻;

provided that each P comprises at least one pair of geminal T¹ and T²wherein T¹ is S and T² is S⁻;

T³ is a group

wherein

indicates the point of attachment to L and wherein

indicates the point of attachment to the rest of the oligonucleotide;

Z is O or S;

U^(5′) is —H or halogen;

R^(5′) is —H;

R^(c1) is —H;

R^(g1), R^(g2), R^(g3), and R^(g4) are H;

R^(3′) is methoxy;

R¹ is —(CH₂)₃—OH;

R² is -methyl; and

n is 1,

wherein Ab is an antibody or antigen-binding fragment thereof that bindsa tumor associated antigen.

In some embodiments, the antibody or conjugate specifically binds anantigen expressed by the cancer or cancer-associated stroma.

In another aspect, also provided herein is a pharmaceutical compositioncomprising a conjugate as described herein. In still another aspect,provided herein is a pharmaceutical composition comprising animmunomodulating oligonucleotide as described herein. In still yet afurther aspect, provided herein is a kit comprising a conjugate asdescribed herein, and instructions for use of the conjugate.

In still further aspects, provided herein are methods for preparing theconjugates as described herein. In one aspect, provided herein is amethod for preparing a conjugate that comprises an antibody orantigen-binding fragment thereof (Ab) and one or more immunomodulatingoligonucleotides (P), wherein the antibody or antigen-binding fragmentis linked to one or more Q-tag peptides (Q) comprising the amino acidsequence RPQGF (SEQ ID NO:47), wherein each immunomodulatingoligonucleotide is linked to a Q-tag peptide via an amide bond with theglutamine residue of the Q-tag peptide and a linker (L) as shown informula (A),

wherein:

indicates the point of attachment of each Q to the antibody orantigen-binding fragment thereof (Ab);

each Q independently comprises a Q-tag peptide sequence RPQGF (SEQ IDNO:47);

each L is independently a bond or a linker moiety connected to Q via anamide bond with the glutamine residue; and

each P is independently an immunomodulating oligonucleotide;

comprising contacting a compound of formula (B)

wherein Ab and Q are as defined for formula (A) above, and e is aninteger from 1 to 20, with one or more immunomodulating oligonucleotidesP, wherein each P independently has the following formula:

wherein

X^(5′) is a 5′ terminal nucleoside;

X^(3′) is a 3′ terminal nucleoside;

Y^(PTE) is an internucleoside phosphotriester;

Y^(3′) is a terminal phosphotriester;

each X^(N) is independently a nucleoside;

each Y^(N) is independently an internucleoside linker;

b and c are each independently an integer from 1 to 25; with the provisothat the sum of b and c is at least 5; and

L is a linker moiety having a terminal amine,

in the presence of a transglutaminase.

In another aspect, provided herein is a method for preparing a conjugatethat comprises an antibody or antigen-binding fragment thereof (Ab) andone or more immunomodulating oligonucleotides (P), wherein the antibodyor antigen-binding fragment is linked to one or more Q-tag peptides (Q)comprising at least one glutamine residue, wherein each immunomodulatingoligonucleotide is linked to a Q-tag peptide via an amide bond with theglutamine residue of the Q-tag peptide and a linker (L) as shown informula (A),

wherein:

indicates the point of attachment of each Q to the antibody orantigen-binding fragment thereof (Ab);each Q is independently a Q-tag peptide having at least one glutamineresidue;each L is independently a bond or a linker moiety connected to Q via anamide bond with the glutamine residue; andeach P is independently an immunomodulating oligonucleotide;comprising contacting a compound of formula (B)

wherein Ab and Q are as defined for formula (A) above, and e is aninteger from 1 to 20, with one or more immunomodulating oligonucleotidesP, wherein each immunomodulating oligonucleotide P is independently anoligonucleotide of formula (C) as described herein or an oligonucleotideof formula (D) according to as described herein, in the presence of atransglutaminase. In some embodiments of the present aspects of methodsof preparing conjugates, each immunomodulating oligonucleotide isindependently an oligonucleotide of formula (C) or formula (D) isselected from the group consisting of the oligonucleotides of Table 10and Table 12.

In still other aspects, provided herein is an antibody or antigenbinding fragment thereof that binds to CD22, wherein the antibody orfragment comprises a heavy chain variable (VH) domain and a light chainvariable (VL) domain, wherein the VH domain comprises a VH domainsequence selected from the group consisting of SEQ ID Nos:64-67; andwherein the VL domain comprises a VL domain sequence selected from thegroup consisting of SEQ ID Nos:68-91. In still other aspects, providedherein is an antibody or antigen binding fragment thereof that binds toCD22, wherein the antibody or fragment comprises a heavy chain variable(VH) domain and a light chain variable (VL) domain, wherein the VHdomain comprises a CDR-H1, CDR-H2, and CDR-H3 from a VH domain shown inTable 8, and wherein the VL domain comprises a CDR-L1, CDR-L2, andCDR-L3 from a VL domain shown in Table 8. In some embodiments, theantibody is a Fab, F(ab′)2, Fab′-SH, Fv, scFv, single domain, singleheavy chain, or single light chain antibody or antibody fragment. Insome embodiments, the antibody comprises an Fc region. In someembodiments, the Fc region is a human Fc region selected from the groupconsisting of an IgG1 Fc region, an IgG2 Fc region, and an IgG4 Fcregion. In some embodiments, the Fc region is a wild-type human IgG1,IgG2, or IgG4 Fc region. In some embodiments, the Fc region is a humanFc region comprising one or more amino acid substitutions that reducebinding to C1q. In some embodiments, the Fc region is a human Fc regioncomprising one or more amino acid substitutions that reduce effectorfunction, as compared with a human Fc region that lacks the amino acidsubstitution(s). In some embodiments, the Fc region is: (a) a human IgG1Fc region comprising L234A, L235A, and/or G237A substitutions, aminoacid position numbering according to EU index; (b) a human IgG2 Fcregion comprising A330S and/or P331S substitutions, amino acid positionnumbering according to EU index; or (c) a human IgG4 Fc regioncomprising S228P and/or L235E substitutions, amino acid positionnumbering according to EU index. In some embodiments, the Fc regionfurther comprises an N297A substitution, amino acid position numberingaccording to EU index. In some embodiments, the antibody furthercomprises an amino acid sequence selected from the group consisting ofSEQ ID Nos:92-110.

In still other aspects, provided herein is an antibody that binds tohuman CD22, wherein the antibody comprises an antibody heavy chain andan antibody light chain, wherein the antibody heavy chain comprises thesequence of SEQ ID NO:179 or 180, and the antibody light chain comprisesthe sequence of SEQ ID NO:181 or 182. In some embodiments, the antibodyheavy chain comprises the sequence of SEQ ID NO:179, and the antibodylight chain comprises the sequence of SEQ ID NO:181. In someembodiments, the antibody heavy chain comprises the sequence of SEQ IDNO:179, and the antibody light chain comprises the sequence of SEQ IDNO:182. In some embodiments, the antibody heavy chain comprises thesequence of SEQ ID NO:180, and the antibody light chain comprises thesequence of SEQ ID NO:181. In some embodiments, the antibody heavy chaincomprises the sequence of SEQ ID NO: 180, and the antibody light chaincomprises the sequence of SEQ ID NO:182.

In still other aspects, provided herein is an antibody that binds tohuman Her2, wherein the antibody comprises an antibody heavy chain andan antibody light chain, wherein the antibody heavy chain comprises thesequence of SEQ ID NO:183, 184, 186, or 187, and the antibody lightchain comprises the sequence of SEQ ID NO: 185 or 188. In someembodiments, the antibody heavy chain comprises the sequence of SEQ IDNO:183, and the antibody light chain comprises the sequence of SEQ IDNO:185. In some embodiments, the antibody heavy chain comprises thesequence of SEQ ID NO:184, and the antibody light chain comprises thesequence of SEQ ID NO:185. In some embodiments, the antibody heavy chaincomprises the sequence of SEQ ID NO:186, and the antibody light chaincomprises the sequence of SEQ ID NO:185. In some embodiments, theantibody heavy chain comprises the sequence of SEQ ID NO:187, and theantibody light chain comprises the sequence of SEQ ID NO:185. In someembodiments, the antibody heavy chain comprises the sequence of SEQ IDNO:183, and the antibody light chain comprises the sequence of SEQ IDNO:188. In some embodiments, the antibody heavy chain comprises thesequence of SEQ ID NO:184, and the antibody light chain comprises thesequence of SEQ ID NO:188. In some embodiments, the antibody heavy chaincomprises the sequence of SEQ ID NO:186, and the antibody light chaincomprises the sequence of SEQ ID NO:188. In some embodiments, theantibody heavy chain comprises the sequence of SEQ ID NO:187, and theantibody light chain comprises the sequence of SEQ ID NO:188.

In still other aspects, provided herein is a pharmaceutical compositioncomprising the conjugate or antibody according to any one of theembodiments herein and a pharmaceutically acceptable carrier. In stillother aspect, provided herein is a pharmaceutical composition comprisingthe immunomodulating oligonucleotide according to any one of theembodiments herein and a pharmaceutically acceptable carrier. In stillother aspect, provided herein are methods for treatment of a disease ordisorder. In one aspect, provided herein is a method of treating cancer,comprising administering to an individual an effective amount of theconjugate, immunomodulating oligonucleotide, antibody, or pharmaceuticalcomposition according to any one of the embodiments herein. In oneaspect, provided herein is a method for treating cancer, comprisingadministering to an individual an effective amount of: (a) an immunecheckpoint inhibitor and (b) the conjugate according to any one of theembodiments herein, or the pharmaceutical composition according to anyone of the embodiments herein; wherein the cancer is refractory orresistant to the immune checkpoint inhibitor when administered in theabsence of the conjugate; and wherein the antibody or antigen-bindingfragment thereof binds to human CD22. In some embodiments, the immunecheckpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor. In someembodiments, the immune checkpoint inhibitor is an anti-PD-1 antibody oran anti-PD-L1 antibody. In some embodiments, the administration resultsin reduced growth, size, and/or volume of the cancer. In one aspect,provided herein is a method for treating cancer, comprisingadministering to an individual an effective amount of the conjugateaccording to any one of the embodiments herein, or the pharmaceuticalcomposition according to any one of the embodiments herein; wherein theadministration results in B cell activation in the individual. In oneaspect, provided herein is a method for treating cancer, comprisingadministering to an individual an effective amount of the conjugateaccording to any one of the embodiments herein, or the pharmaceuticalcomposition according to any one of the embodiments herein; wherein thecancer is selected from the group consisting of head and neck squamouscell carcinoma (HNSCC), non-small-cell lung carcinoma (NSCLC), renalcell carcinoma (RCC), gastric cancer, hepatocellular carcinoma (HCC),esophageal cancer, cervical cancer, cervical squamous cell carcinoma,Merkle cell carcinoma, endometrial cancer, ovarian cancer, pancreaticcancer, melanoma, cutaneous melanoma, sarcoma, colorectal cancer, breastcancer, small cell lung cancer (SCLC), cutaneous squamous cellcarcinoma, and urothelial carcinoma. In one aspect, provided herein is amethod for treating cancer, comprising administering to an individual aneffective amount of the conjugate according to any one of theembodiments herein, or the pharmaceutical composition according to anyone of the embodiments herein; wherein the cancer is selected from thegroup consisting of acute lymphoblastic leukemia (ALL), hairy cellleukemia, and diffuse large B cell lymphoma (DLBCL). In one aspect,provided herein is a method for treating cancer, comprisingadministering to an individual an effective amount of the conjugateaccording to any one of the embodiments herein, or the pharmaceuticalcomposition according to any one of the embodiments herein; wherein thecancer is selected from the group consisting of breast cancer,urothelial cancer, and gastric cancer. In some embodiments, theadministration results in reduced growth, size, and/or volume of thecancer. In another aspect, provided herein is the conjugate,immunomodulating oligonucleotide, antibody, or pharmaceuticalcomposition according to any one of the embodiments herein for use in amethod of treating cancer, wherein the method comprises administering aneffective amount of the conjugate, immunomodulating oligonucleotide,antibody, or pharmaceutical composition to an individual. In someembodiments, the cancer is a solid tumor. In some embodiments, thecancer is a liquid tumor. In some embodiments, the cancer is a B cellcancer. In some embodiments, the cancer is a lymphoma or leukemia. Insome embodiments, the cancer is breast cancer, colorectal cancer, lungcancer, head and neck cancer, melanoma, lymphoma, or leukemia. In stillother aspect, provided herein is a method for delivering theimmunomodulating oligonucleotide according to any of the embodimentsherein, comprising contacting the immunomodulating oligonucleotide witha cell. In some embodiments, the immunomodulating oligonucleotide ispegylated. In some embodiments, the immunomodulating oligonucleotide isformulated in a nanoparticle. In some embodiments, the immunomodulatingoligonucleotide is conjugated to a polypeptide. In some embodiments, theantibody or conjugate specifically binds an antigen expressed by thecancer or cancer-associated stroma.

DESCRIPTION OF THE FIGURES

The present application can be understood by reference to the followingdescription taken in conjunction with the accompanying figures.

FIGS. 1A and 1B depict the activity of immunomodulating oligonucleotidesalone in human PBMCs based upon observed increased expression of (a)HLADR and (b) CD40.

FIGS. 2A-2D show the effect of immunomodulating polynucleotides andtheir antibody conjugates on increasing B cells numbers and activation.FIG. 2A depicts the observed effect on B cell numbers by the variousimmunomodulating polypeptides alone. FIGS. 2B-2C depict the observedactivation of B cells (via detection of CD40 expression) produced by theimmunomodulating polynucleotides alone. FIG. 2D depicts the observedactivity of antibody-oligonucleotide conjugates with RFB4 (anti-CD22)(DAR1).

FIG. 3 shows the percentage yields of transglutaminase-mediatedconjugations and deconjugations with a polyethylene glycol linker(—NH—C(═O)—PEG₂₃-NH₂) and various Q-tags. Shown are SEQ ID Nos:39-47 and49-52.

FIGS. 4A and 4B show the percentage change of conjugation anddeconjugation over time in transglutaminase conjugation of two Q-tagpeptides (LSLSPGLLQGG, SEQ ID NO:39; and RPQGF, SEQ ID NO:47).

FIGS. 5A-5D depict activity of RBF4 conjugates with variousoligonucleotides as shown by their B-cell activation, as assessed byexpression of (a) CD40, (b) CD86, (c) HLADR and (d) CD69.

FIGS. 6A-6D depict activity of BDCA2 conjugates with variousoligonucleotides as shown by their pDC activation, as assessed byexpression of (a) CD86, (b) CD40, (c) HLADR, or (d) IFNα.

FIGS. 7A-7D show the activity of BDCA2 conjugates with variousoligonucleotides as shown by their activation of (a) monocytes; (b)mDCs; (c) CD19⁺ B cells; or (d) CD3⁺ T cells.

FIG. 8A shows an alignment of mouse and humanized anti-CD22 variableheavy (VH) domain sequences. CDR-H1, -H2, and -H3 are depicted in boxes.Sequences correspond to SEQ ID Nos:56 and 64-67 (top to bottom).

FIG. 8B shows an alignment of mouse and humanized anti-CD22 variablelight (VL) domain sequences. CDR-L1, -L2, and -L3 are depicted in boxes.Sequences correspond to SEQ ID Nos:57 and 68-72 (top to bottom).

FIG. 9 shows relative expression levels of each combination of humanizedanti-CD22 heavy chain and light chain, as indicated.

FIGS. 10A-10E show purification of antibody:CpG conjugates. Shown arethe purification of conjugates using antibodies TNT70 (FIG. 10A), TNT71(FIG. 10B), TNT72 (FIG. 10C), and TNT74 (FIG. 10D). FIG. 10E showsanalysis of starting material and CpG conjugates in reduced (r) ornon-reduced (n.r.) form, as indicated.

FIG. 11 shows B cell activation by various humanized anti-CD22antibody:CpG conjugates at different concentrations, as indicated.

FIGS. 12A-12D show profiles of the indicated CpG:anti-CD22 conjugates,as determined by size exclusion chromatography HPLC (HPLC-SEC).Conjugates using the RH1 (FIG. 12A), RH2 (FIG. 12B), RH3 (FIG. 12C), andRH4 (FIG. 12D) VH domains are grouped.

FIG. 13A summarizes selected properties of the indicated CpG:anti-CD22conjugates, including expression level in Expi293 or CHO cells, % highmolecular weight (HMW) or monomer species by SEC-HPLC, VH/VL pairing,binding affinity to human CD22, and binding to cynomolgus CD22. FIG. 13Bshows expression level of the indicated CpG:anti-CD22 conjugates fromCHO cells.

FIG. 14 shows results from analysis of thermal stability of theindicated antibodies and antibody conjugates by differential scanningcalorimetry (DSC).

FIGS. 15A and 15B show the expression of N92 substitution variants ofthe RL1 VL domain under non-reducing (FIG. 15A) or reducing (FIG. 15B)conditions.

FIGS. 15C-15F show the binding kinetics of antibodies including the N92substitution variants of the RL1 VL domain to CD22, including KD (FIG.15C), Ka (FIG. 15D), Kd (FIG. 15E), and capture levels (FIG. 15F), asdetermined by SPR.

FIGS. 16A-16E show a schematic diagram of exemplary conjugates, inaccordance with some embodiments. Exemplary antibody:CpG conjugates withan engineered Q-tag (RPQGF; SEQ ID NO:47) fused to the C-terminus of theheavy chain are shown in FIGS. 16A & 16E (with a DAR 1) and in FIG. 16B(with DAR 2). Exemplary antibody:CpG conjugates with an naturallyoccurring Q-tag (Q295) exposed for conjugation by an N297A mutation areshown in FIG. 16C (with a DAR 1) and in FIG. 16D (with DAR 2).

FIGS. 17A-17B depict pharmacokinetic properties of CpG-antibodyconjugates. FIG. 17A shows the half-life of CpG-antibody conjugates ofCmpd 1.1b (SEQ ID NO:3), Cmpd 3.2b (SEQ ID NO:9), Cmpd 4.2b (SEQ IDNO:12), Cmpd 4.3b (SEQ ID NO:13), Cmpd 5.2a (SEQ ID NO:15) or Cmpd 5.7a(SEQ ID NO:20) with RFB4 (SEQ ID NOS: 56 and 57) as compared to nakedRFB4. FIG. 17B shows the half-life of RFB4 conjugates as evaluated bycapturing the 5′ region of the CpG using anti-BrdU antibody; conjugatesof RFB4 with Cmpd 3.2b (SEQ ID NO:9), Cmpd 4.2b (SEQ ID NO: 12), Cmpd4.3b (SEQ ID NO:13, Cmpd 5.2a (SEQ ID NO:15) and Cmpd 5.7a (SEQ IDNO:20) have increased half-life compared to the RFB4 conjugate with Cmpd1.1b (SEQ ID NO:3).

FIG. 18 shows activation of B cells from cynomolgus monkey PBMCs by CpGoligonucleotides, as measured by CD86 expression. Treatment withcompound 7.7b (7-7) induced superior CD86 upregulation as compared tothat induced by compound 1.1b (12070), demonstrating enhanced B cellactivation.

FIG. 19 shows IL-6 induction following treatment with anti-CD22:CpGoligonucleotide conjugate treatment of human PBMCs. Anti-CD22 conjugatedto compound 1.1b (12070) CpG induced IL6 expression not achieved byunconjugated anti-CD22 antibody.

FIGS. 20A & 20B show non-targeted activation of T cells (FIG. 20A) and Bcells (FIG. 20B) by anti-SIRP-α conjugated to compound 1.1b (12070), ascompared to treatment with unconjugated anti-SIRP-α.

FIGS. 21A-21C show results of tumor immune rechallenge assay in mice.MC38 cells were injected into the right flank of C57BL/6 female mice, ata concentration of 2×10⁶ cells per mouse in DMEM. Tumors were monitoreduntil the average size of tumors reached 150-155 mm³. Mice wererandomized into PBS control, anti-SIRP-α-4523 CpG or anti-mCD22-4523 CpGat 5 mice per cohort. Anti-SIRP-α-4523 and anti-mCD22-4523 were dosed at10 mg/kg two times in total, three days apart. Both drugs wereadministered intraperitoneally. On day 88, mice with eradicated tumorswere re-challenged with MC38 (left flank) at 2×10⁶ cells per mouse inDMEM. Naïve mice that have not been implanted with MC38 mice wereincluded as control for tumor growth. Tumors were measured in twodimensions with calipers, and tumor volume was calculated as:length×width×width×0.5, where length was the larger of the twomeasurements. Following re-challenge, previously eradicated mice showedefficient tumor rejection as early as three days post-tumorre-implantation suggesting that treatment with both anti-SIRP-α (FIG.21B) and anti-mCD22 (FIG. 21C) conjugated to 4523 CpG elicited robustimmune memory response against the implanted tumor, not seen in naïvemice who are encountering MC38 tumor for the first time (FIG. 21A).

FIGS. 22A-22D show that an active Fc region (FIGS. 22A & 22C) issuperior for targeting tumors with anti-Her2:CpG oligonucleotideconjugates, as compared to less active Fc region (FIGS. 22B & 22D). m/hHer2 expressing MC38 cells was generated by lentiviral transduction andsorted to obtain cells that express m/h Her2. m/h Her2-MC38 cells wereinjected into the right flank of C57BL/6 female mice, at a concentrationof 2×10⁶ cells per mouse in DMEM. Tumors were monitored until theaverage size of tumors reached 70 mm³. Mice were randomized into PBScontrol, TNT149a blocking (anti-Her2 mIgG2a), and TNT150a (anti-Her2mIgG1) with 5 mice per cohort. Anti-Her2-CpG nucleotideconjugate-treated mice were dosed with 1, 3 and 10 mg/kg three times intotal, three days apart (FIGS. 22A & 22B). Both drugs were administeredintraperitoneally. Arrows indicate administration. By day 60, 1, 3 and10 mg/kg TNT149a treated mice dosed three times, three days apart showedtumor eradication (1/5, 5/5 and 5/5 mice, respectively; FIG. 22C) whilemice treated with 1, 3 and 10 mg/kg TNT150a showed lower number of micewith eradicated tumors (0/5, 3/5 and 5/5, respectively; FIG. 22D). Micetreated with PBS control reached endpoint by day 24 and all groupstreated with TNT149a or TNT150a showed delayed tumor growth as comparedto PBS control.

FIGS. 23A-23I show the results of an MC38 m/h Her2 mouse immunere-challenge assay. FIG. 23A shows a diagram of the assay. On day 81,mice with eradicated tumors were rechallenged with m/h Her2 MC38 (lowerright flank), parent MC38 (lower left flank), m/h Her2 B16F10 (upperright flank) and parent B16F10 (upper left flank) at 2×10⁶ cells (m/hHer2 MC38 and MC38 cells) and 1×10⁶ cells (m/h Her2 B16F10 and B16F10cells) per mouse in DMEM. On day 81, there were 5 mice with eradicatedtumors for groups treated with 10 mg/kg TNT150a, 3 and 10 mg/kg TNT149aand 3 mice with eradicated tumors for group treated with 3 mg/kgTNT150a. Naïve mice showed growth for all implanted cells. m/h Her2B16F10, MC38 and m/h Her2 MC38 showed eradicated tumors or significantdelayed tumor growth as compared to naïve. In all groups, the parentB16F10 tumors grew with one mouse that showed no growth in the 10 mg/kgmIgG2a group. By day 99, all mice showed complete eradication with m/hHer2 MC38 cells. Both MC38 parent and m/h Her2 B16F10 cells showed tumoreradication with the exception of one mouse for both 3 and 10 mg/kg micepreviously treated with TNT149a for MC38 parent cells and 1-3 mice forall previously 3 and 10 mg/kg treated groups. These data show that m/hHer2 MC38 tumor bearing mice with eradicated tumors after treatment withanti-Her2 mIgG1 and mIgG2a have potent and durable anti-tumor responseto m/h Her2 MC38, parent MC38 and m/h B16F10 but not parent B16F10tumors. FIGS. 23B-23I show the results of the individual mice in there-challenge groups, B16F10, MC38, m/h Her2 B6F10 and m/h Her2 MC38tumors respectively.

FIGS. 24A-24C show the results of NanoString gene expression analysis in8 hours after IP administration of anti-mCD22:CpG oligo conjugates.Normalized expression data of the tumors were analyzed with nSolver andnCounter Advanced Analysis Software to obtain gene expression signaturesas defined by NanoString. CT26-bearing mice treated with anti-mCD22 CpGshowed higher signature scores for interferon signaling (FIG. 24A),antigen presentation (FIG. 24B), and cytotoxicity (FIG. 24C) in thetumor as compared to PBS control and anti-mCD22 alone.

FIG. 25 shows the results of co-culturing murine bone marrow-derivedmacrophages with Her2-positive or Her2-negative tumor cells in thepresence of anti-Her2:CpG conjugates with active or inactive Fc regions.

FIGS. 26A-26C show induction of B cell activation in human PBMCs byanti-CD22:CpG oligonucleotide conjugates, as compared to naked CpGs.Shown are expression of CD40 (FIG. 26A), CD80 (FIG. 26B), and CD86 (FIG.26C) following treatment of human PBMCs with anti-CD22 antibody with RH2VH domain and RL1 N92S VL domain (SEQ ID Nos:65 and 87, respectively)conjugated to compound 7.7b (7-7) CpG, TNT52a (RFB4 conjugated to12070), compound 7.7b (7-7) CpG, 12070 CpG, or media only.

FIG. 27 shows activity of indicated free CpG oligonucleotides on humanPBMCs, as assayed by CD40 expression on CD19+ B cells.

FIG. 28 shows activity of indicated free CpG oligonucleotides on humanPBMCs, as assayed by Ramos NFkb Reporter Assay.

FIGS. 29A-29C show activity of indicated free CpG oligonucleotides onhuman PBMCs from three different donor lines (D559, D804 and D643), asobserved by CD40 expression.

FIG. 30 shows activity of indicated free CpG oligonucleotides on humanPBMCs, as assayed by CD40 expression on CD19+ B cells.

FIG. 31 shows activity of CpG:antibody conjugates on human PBMCs, asassayed by CD40 expression on CD19+ B cells.

FIGS. 32A-32C show activity of indicated anti-CD22 antibody:CpGoligonucleotide conjugates on human PBMCs, as assayed by CD40 expressionon CD19+ B cells.

FIG. 33 shows tumor volume over time after treatment with anti-CD22antibody:CpG oligonucleotide conjugate having a “dead” Fe domain at theindicated number of doses, as compared to PBS. Tumor bearing syngeneicmodel using CT26 mouse colon carcinoma cells was used. The conjugateswere administered intravenously 1 to 3 doses every 3 days at 10 mg/kg.

FIGS. 34A-34D show effects of treatment with unconjugated CpGoligonucleotide, unconjugated anti-CD22 antibody, or anti-CD22antibody:CpG oligonucleotide conjugate on different types of immunecells. Effects on B cells were assayed using the CD40 activation marker(FIG. 34A), effects on dendritic cells were assayed using the CD80activation marker (FIG. 34B), effects on CD14+ myeloid cells wereassayed using the CD86 activation marker (FIG. 34C), and effects on Tcells were assayed using the CD69 activation marker (FIG. 34D).

FIGS. 35A & 35B show activation of monocytes, macrophages, and dendriticcells upon treatment with anti-huHer2 antibody having mIgG1 or mIgG2a Fcdomain, either unconjugated or conjugated to mouse CpG oligonucleotide.FIG. 35A shows co-culture of splenocytes from Balbc syngeneic mouse(full immune system) in the presence of human breast tumor cell lineSKBR3 (Her2+++), gating on monocytes/macrophages (Lin-CD11b+,F480+,GR1mid), and assaying CD40 expression. FIG. 35B shows co-cultureof splenocytes from Balbc syngeneic mouse (full immune system) in thepresence of human breast tumor cell line SKBR3 (Her2+++), gating ondendritic cells (lin−, F480−, CD11c+, MHCII+), and assaying CD40expression.

FIG. 36 shows anti-tumor efficacy of anti-CD22 antibody conjugated toCpG oligonucleotide according to 3 dosing regimens in EMT6 breast cancermodel that is generally refractory to treatment with anti-PD1/PD-L1.

FIG. 37 shows anti-tumor efficacy of anti-CD22 antibody conjugated toCpG oligonucleotide or free CpG oligonucleotide in EMT6 breast cancermodel when administered intratumorally.

FIGS. 38A-38D show an increase in B cell differentiation and decrease inB regulatory cells in the spleen following administration of anti-CD22Ab-CpG conjugate.

FIGS. 39A-39D show an increase in CD4 and CD8 T effector cells andfunction in the spleen following administration of anti-CD22 Ab-CpGconjugate.

FIGS. 40A-40D show an increase in B cell infiltrates and modulation ofsuppressive microenvironment in the tumor following administration ofanti-CD22 Ab-CpG conjugate.

FIGS. 41A-41C show robust induction of cytokines and chemokines uponCD22-mediated TLR9 engagement in human B cells.

FIGS. 42A & 42B show robust induction of various cytokines andchemokines (as indicated) with anti-mCD22-CpG with no apparentaccumulation in the periphery upon repeat dosing in mouse.

FIG. 43 shows anti-tumor response in groups of CT26 syngeneic mice thatare non-responders to anti-PD1 antibody treatment. The treatment isperformed using an anti-mouse CD22 antibody (SEQ ID NO: 124 and SEQ IDNO: 125) conjugated to mouse CpG 4523 (SEQ ID NO: 121). Tumor volume wasreduced in mice treated with the conjugate or a combination of theconjugate and anti-PD1 antibody, whereas respective mice treated withonly anti-PD1 antibody did not experience a significant reduction intumor volume.

FIG. 44 shows effect of treatment with an anti-mouse CD22 antibody (SEQID NO: 124 and SEQ ID NO: 125) conjugated to mouse CpG 4523 (SEQ ID NO:121) with a DAR1 configuration in BALB/C mice implanted with 4T1 cells.The number of metastatic plaques was significantly reduced in the CD22Ab-CpG conjugate as compared to the control.

DETAILED DESCRIPTION

The following description sets forth exemplary methods, parameters andthe like. It should be recognized, however, that such description is notintended as a limitation on the scope of the present disclosure but isinstead provided as a description of exemplary embodiments.

The present description is based on the discovery that certainpolypeptide-oligonucleotide conjugates provide enhanced stability anddelivery selectivity. The description also provides the methods forpreparing these conjugates. Particularly, the conjugation can beperformed by a transglutaminase (Tgase)-mediated reaction. Thedescription also provides intermediate compounds that can be used toprepare these conjugates as well as compositions and kits that containthese polypeptide-oligonucleotide conjugates.

I. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which this disclosure belongs. All patents, applications,published applications and other publications referred to herein areincorporated by reference in their entireties. If a definition set forthin this section is contrary to or otherwise inconsistent with adefinition set forth in a patent, application, or other publication thatis herein incorporated by reference, the definition set forth in thissection prevails over the definition incorporated herein by reference.

It is appreciated that certain features of the disclosure, which are,for clarity, described in the context of separate embodiments, may alsobe provided in combination in a single embodiment. Conversely, variousfeatures of the disclosure, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination. All combinations of the embodimentspertaining to particular method steps, reagents, or conditions arespecifically embraced by the present disclosure and are disclosed hereinjust as if each and every combination was individually and explicitlydisclosed.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural referents unless the context clearly dictatesotherwise. It is further noted that the claims may be drafted to excludeany optional element. As such, this statement is intended to serve asantecedent basis for use of such exclusive terminology as “solely,”“only” and the like in connection with the recitation of claim elements,or use of a “negative” limitation.

Throughout this application, unless the context indicates otherwise,references to a compound of Formula (A)-(D) include ionic forms,polymorphs, pseudopolymorphs, amorphous forms, solvates, co-crystals,chelates, isomers, tautomers, oxides (e.g., N-oxides, S-oxides), esters,prodrugs, isotopes and/or protected forms thereof. In some embodiments,references to a compound of Formula (A)-(D) include polymorphs,solvates, co-crystals, isomers, tautomers and/or oxides thereof. In someembodiments, references to a compound of Formula (A)-(D) includepolymorphs, solvates, and/or co-crystals thereof. In some embodiments,references to a compound of Formula (A)-(D) include isomers, tautomersand/or oxides thereof. In some embodiments, references to a compound ofFormula (A)-(D) include solvates thereof.

“Alkyl” encompasses straight and branched carbon chains having theindicated number of carbon atoms, for example, from 1 to 20 carbonatoms, or 1 to 8 carbon atoms, or 1 to 6 carbon atoms. For example, C₁₋₆alkyl encompasses both straight and branched chain alkyl of from 1 to 6carbon atoms. When an alkyl residue having a specific number of carbonsis named, all branched and straight chain versions having that number ofcarbons are intended to be encompassed; thus, for example, “propyl”includes n-propyl and isopropyl; and “butyl” includes n-butyl,sec-butyl, isobutyl and t-butyl. Examples of alkyl groups include, butare not limited to, methyl, ethyl, propyl, isopropyl, n-butyl,sec-butyl, tert-butyl, pentyl, 2-pentyl, 3-pentyl, isopentyl, neopentyl,hexyl, 2-hexyl, 3-hexyl, and 3-methylpentyl.

When a range of values is given (e.g., C₁₋₆ alkyl), each value withinthe range as well as all intervening ranges are included. For example,“C₁₋₆ alkyl” includes C₁, C₂, C₃, C₄, C₅, C₆, C₁₋₆, C₂₋₆, C₃₋₆, C₄₋₆,C₅₋₆, C₁₋₅, C₂₋₅, C₃₋₅, C₄₋₅, C₁₋₄, C₂₋₄, C₃₋₄, C₁₋₃, C₂₋₃, and C₁₋₂alkyl.

“Alkenyl” refers to an unsaturated branched or straight-chain alkylgroup having the indicated number of carbon atoms (e.g., 2 to 8, or 2 to6 carbon atoms) and at least one carbon-carbon double bond. The groupmay be in either the cis or trans configuration (Z or E configuration)about the double bond(s). Alkenyl groups include, but are not limitedto, ethenyl, propenyl (e.g., prop-1-en-1-yl, prop-1-en-2-yl,prop-2-en-1-yl (allyl), prop-2-en-2-yl), and butenyl (e.g.,but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl,but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl).

“Alkynyl” refers to an unsaturated branched or straight-chain alkylgroup having the indicated number of carbon atoms (e.g., 2 to 8 or 2 to6 carbon atoms) and at least one carbon-carbon triple bond. Alkynylgroups include, but are not limited to, ethynyl, propynyl (e.g.,prop-1-yn-1-yl, prop-2-yn-1-yl) and butynyl (e.g., but-1-yn-1-yl,but-1-yn-3-yl, but-3-yn-1-yl).

The term “amino,” as used herein, represents —N(R^(N1))₂, where, ifamino is unsubstituted, both R^(N1) are H; or, if amino is substituted,each R^(N1) is independently H, —OH, —NO₂, —N(R^(N2))₂, —SO₂OR^(N2),—SO₂R^(N2), —SOR^(N2), —COOR^(N2), an N-protecting group, alkyl,alkenyl, alkynyl, alkoxy, aryl, arylalkyl, aryloxy, cycloalkyl,cycloalkenyl, heteroalkyl, or heterocyclyl, provided that at least oneR^(N1) is not H, and where each R^(N2) is independently H, alkyl, oraryl. Each of the substituents may itself be unsubstituted orsubstituted with unsubstituted substituent(s) defined herein for eachrespective group. In some embodiments, amino is unsubstituted amino(i.e., —NH₂) or substituted amino (e.g., —NHR^(N1)), where R^(N1) isindependently —OH, —SO₂OR^(N2), —SO₂R^(N2), —SOR^(N2), —COOR^(N2),optionally substituted alkyl, or optionally substituted aryl, and eachR^(N2) can be optionally substituted alkyl or optionally substitutedaryl. In some embodiments, substituted amino may be alkylamino, in whichthe alkyl groups are optionally substituted as described herein foralkyl. In certain embodiments, an amino group is —NHR^(N1), in whichR^(N1) is optionally substituted alkyl. Non-limiting examples of—NHR^(N1), in which R^(N1) is optionally substituted alkyl, include:optionally substituted alkylamino, a proteinogenic amino acid, anon-proteinogenic amino acid, a C₁₋₆ alkyl ester of a proteinogenicamino acid, and a C₁₋₆ alkyl ester of a non-proteinogenic amino acid.The amino acid employed is optionally in the L-form.

The term “immunomodulating polynucleotide” as used herein, represents apolynucleotide construct containing a total of from 6 to 50 contiguousnucleosides covalently bound together by internucleoside bridging groupsindependently selected from the group consisting of internucleosidephosphoesters and optionally internucleoside abasic spacers. Theimmunomodulating polynucleotides are capped at 5′- and 3′-termini with5′- and 3′-capping groups, respectively. The immunomodulatingpolynucleotides are capable of modulating an innate immune response, asdetermined by, e.g., a change in the activation of intracellularsignaling pathway(s) including but not limited to NFκB, a change in theexpression of an activation marker or a change in the secretion of atleast one inflammatory cytokine or at least one type I interferon in animmune cell (e.g., antigen-presenting cell) to which an immunomodulatingpolynucleotide was delivered (e.g., in comparison to another immune cell(e.g., antigen-presenting cell) to which an immunomodulatingpolynucleotide was not delivered) or in an immune cell that interactswith an immune cell (e.g., antigen-presenting cell) to which animmunomodulating polynucleotide was delivered (including directcell-to-cell interactions as well as indirect stimulation, e.g., fromone or more cytokines secreted by the cell to which an immunomodulatingpolynucleotide was delivered). The immunomodulating polynucleotide maycontain a conjugating group or, if the immunomodulating polynucleotideis part of a conjugate, a linker bonded to a targeting moiety andoptionally to one or more (e.g., 1 to 6) auxiliary moieties (e.g.,polyethylene glycols). The conjugating group or the linker may be partof the phosphotriester or the terminal capping group.

The term “immunostimulating polynucleotide” as used herein, representsan immunomodulating polynucleotide capable of activating an immuneresponse, as determined by, e.g., an increase in the activation ofintracellular signaling pathway(s) such as NFκB or an increase in levelsof cell surface marker(s) of activation or function or an increase inthe secretion of at least one inflammatory cytokine or at least one typeI interferon in an immune cell (e.g., antigen-presenting cell) to whichan immunostimulating polynucleotide was delivered (e.g., in comparisonto another immune cell (e.g., antigen-presenting cell) to which animmunostimulating polynucleotide was not delivered) or in an immune cellthat interacts with an immune cell (e.g., antigen-presenting cell) towhich an immunomodulating polynucleotide was delivered (including directcell-to-cell interactions as well as indirect stimulation, e.g., fromone or more cytokines secreted by the cell to which an immunomodulatingpolynucleotide was delivered). In some embodiments, theimmunostimulating polynucleotide contains at least onecytidine-p-guanosine (CpG) sequence, in which p is an internucleosidephosphodiester (e.g., phosphate or phosphorothioate) or aninternucleoside phosphotriester or phosphothiotriester. As used herein,the CpG-containing immunostimulating polynucleotide can be naturallyexisting, such as CpG ODNs of bacterial or viral origins, or synthetic.For example, in some embodiments, the CpG sequence in theimmunostimulating polynucleotide contains 2′-deoxyribose. In someembodiments, the CpG sequence in the immunostimulating polynucleotide isunmethylated. In some embodiments, the immunostimulating polynucleotideis a polynucleotide of Formula (C) as provided herein. In someembodiments, the immunostimulating polynucleotide is compound of Formula(D) as provided herein.

The term “immunosuppressive polynucleotide” as used herein, representsan immunomodulating polynucleotide capable of antagonizing an immuneresponse, as determined by e.g., a reduction in the activation or lackof activation of NFκB or lack on increase in the levels of cell surfacemarker(s) of activation of function or a reduction or lack of increasein the secretion of at least one inflammatory cytokine or at least onetype I interferon in an immune cell (e.g., antigen-presenting cell) towhich an immunosuppressive polynucleotide was delivered (e.g., incomparison to another immune cell (e.g., antigen-presenting cell) towhich an immunosuppressive polynucleotide was not delivered) or in animmune cell that interacts with an immune cell (e.g., antigen-presentingcell) to which an immunomodulating polynucleotide was delivered(including direct cell-to-cell interactions as well as indirectstimulation, e.g., from one or more cytokines secreted by the cell towhich an immunomodulating polynucleotide was delivered).

It is to be understood that the terms “polynucleotide” and“oligonucleotide” may be used interchangeably herein. It is further tobe understood that the terms “immunomodulating polynucleotide,”“immunostimulating polynucleotide,” “immunosuppressive polynucleotide,”and “conjugate” encompass salts of the immunomodulating polynucleotide,immunostimulating polynucleotide, immunosuppressive polynucleotide andconjugate, respectively. For example, the terms “immunomodulatingpolynucleotide,” “immunostimulating polynucleotide,” “immunosuppressivepolynucleotide,” and “conjugate” encompasses both the protonated,neutral form (P—XH moiety, where X is O or S) of a phosphate,phosphorothioate, or phosphorodithioate and the deprotonated, ionic form(P—X⁻ moiety, where X is O or S) of a phosphate, phosphorothioate, orphosphorodithioate. Accordingly, it is to be understood that thephosphoesters and phosphodiesters described as having one or more ofR^(E1), R^(E2), and R^(E3) as hydrogen encompass salts, in which thephosphate, phosphorothioate, or phosphorodithioate is present in adeprotonated, ionic form. In addition, the terms “free,” “naked,” and“unconjugated” referring to immunomodulating polynucleotides,immunostimulating polynucleotides, immunosuppressive polynucleotides,and/or oligonucleotides (e.g., CpG oligonucleotides) may be usedinterchangeably herein.

The term “phosphotriester,” as used herein, refers to a phosphoester, inwhich all three valences are substituted with non-hydrogen substituents.The phosphotriester consists of phosphate, phosphorothioate, orphosphorodithioate; one or two bonds to nucleoside(s), or abasicspacer(s), and/or phosphoryl group(s); and one or two groupsindependently selected from the group consisting of a bioreversiblegroup; a non-bioreversible group; an auxiliary moiety; a conjugatinggroup; and a linker bonded to a targeting moiety and optionally to oneor more (e.g., 1 to 6) auxiliary moieties. A terminal phosphotriesterincludes one bond to a group containing a nucleoside and two groupsindependently selected from the group consisting of a bioreversiblegroup; a non-bioreversible group; an auxiliary moiety; a conjugatinggroup; a phosphoryl group; and a linker bonded to a targeting moiety andoptionally to one or more (e.g., 1 to 6) auxiliary moieties. In someembodiments, a terminal phosphotriester contains 1 or 0 linkers bondedto a targeting moiety and optionally to one or more (e.g., 1 to 6)auxiliary moieties. An internucleoside phosphotriester includes twobonds to nucleoside-containing groups. A phosphotriester may be a groupof the following structure:

wherein:

each of X^(E1) and X^(E2) is independently O or S;

each or R^(E1) and R^(E3) is independently a bond to a nucleoside; asugar analogue of an abasic spacer; a bioreversible group; anon-bioreversible group; an auxiliary moiety; a conjugating group; alinker bonded to a targeting moiety; a linker bonded to a targetingmoiety and one or more (e.g., 1 to 6) auxiliary moieties; or thephosphorus atom in a group of formula —P(═X^(E1))(—X^(E2)-R^(E2)A)-O—,

-   -   where R^(E2)A is hydrogen; a bioreversible group; a        non-bioreversible group; an auxiliary moiety; a conjugating        group; a linker bonded to a targeting moiety; or a linker bonded        to a targeting moiety and one or more (e.g., 1 to 6) auxiliary        moieties; and

R^(E2) is a bioreversible group; a non-bioreversible group; an auxiliarymoiety; a conjugating group; a linker bonded to a targeting moiety; or alinker bonded to a targeting moiety and one or more (e.g., 1 to 6)auxiliary moieties;

provided that at least one of R^(E1) and R^(E3) is a bond to a groupcontaining at least one nucleoside.

If both R^(E1) and R^(E3) are bonds to groups containing at least onenucleoside, the phosphotriester is an internucleoside phosphotriester.If one and only one of R^(E1) and R^(E3) is a bond to a group containinga nucleoside, the phosphotriester is a terminal phosphotriester.

As used herein, the term “amino acid” refers to any amino acid (bothstandard and non-standard amino acids), including, but not limited to,α-amino acids, β-amino acids, γ-amino acids and δ-amino acids. Examplesof suitable amino acids include, but are not limited to, alanine,asparagine, aspartate, cysteine, glutamate, glutamine, glycine, proline,serine, tyrosine, arginine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, threonine, tryptophan and valine. Additionalexamples of suitable amino acids include, but are not limited to,omithine, hypusine, 2-aminoisobutyric acid, dehydroalanine,gamma-aminobutyric acid, citrulline, beta-alanine, alpha-ethyl-glycine,alpha-propyl-glycine and norleucine.

The terms “antibody,” “immunoglobulin,” and “Ig” are usedinterchangeably herein, and are used in the broadest sense andspecifically cover, for example, individual monoclonal antibodies(including agonist, antagonist, neutralizing antibodies, full length orintact monoclonal antibodies), antibody compositions with polyepitopicor monoepitopic specificity, polyclonal or monovalent antibodies,multivalent antibodies, multispecific antibodies (e.g., bispecificantibodies so long as they exhibit the desired biological activity),formed from at least two intact antibodies, single chain antibodies, andfragments of antibodies. An antibody can be human, humanized, chimericand/or affinity matured as well as an antibody from other species, forexample, mouse and rabbit.

The term “antibody” is intended to include a polypeptide product of Bcells within the immunoglobulin class of polypeptides that is able tobind to a specific antigen and is composed of two identical pairs ofpolypeptide chains, wherein each pair has one heavy chain (about 50-70kDa) and one light chain (about 25 kDa) and each amino-terminal portionof each chain includes a variable region of about 100 to about 130 ormore amino acids and each carboxyl-terminal portion of each chainincludes a constant region. See Borrebaeck (ed.) (1995) AntibodyEngineering, Second Ed., Oxford University Press.; Kuby (1997)Immunology, Third Ed., W.H. Freeman and Company, New York. Antibodiesalso include, but are not limited to, synthetic antibodies, monoclonalantibodies, recombinant antibodies, multispecific antibodies (includingbi-specific antibodies), human antibodies, humanized antibodies,camelized antibodies, chimeric antibodies, intrabodies, anti-idiotypic(anti-Id) antibodies, and functional fragments thereof, which refers aportion of an antibody heavy or light chain polypeptide that retainssome or all of the binding activity of the antibody from which thefragment is derived. Non-limiting examples of functional fragments of anantibody include single-chain Fvs (scFv) (e.g., including monospecificor bispecific), Fab fragments, F(ab′) fragments, F(ab)₂ fragments,F(ab′)2 fragments, disulfide-linked Fvs (sdFv), Fd fragments, Fvfragments, scRv-Fc, nanobody, diabody, triabody, tetrabody, andminibody. In some embodiments, the antibody comprises an Fc variant thathas reduced or ablated effector function. In particular, antibodiesprovided herein include immunoglobulin molecules and immunologicallyactive portions of immunoglobulin molecules, for example,antigen-binding domains or molecules that contain an antigen-bindingsite that binds to the antigen (e.g., one or more complementaritydetermining regions (CDRs) of an anti-CD56 antibody or an anti-SIRPαantibody). Such antibody fragments are described in, for example, Harlowand Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, New York (1989); Myers (ed.), Molec. Biology andBiotechnology: A Comprehensive Desk Reference, New York: VCH Publisher,Inc.; Huston et al., Cell Biophysics 1993, 22, 189-224; Plückthun andSkerra, Meth. Enzymol. 1989, 178, 497-515; and Day, AdvancedImmunochemistry, Second Ed., Wiley-Liss, Inc., New York, N.Y. (1990).The antibodies provided herein can be of any type (e.g., IgG, IgE, IgM,IgD, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, andIgA2), or any subclass (e.g., IgG2a and IgG2b) of an immunoglobulinmolecule.

The term “antigen” refers to a predetermined target to which an antibodycan selectively bind. A target antigen can be a polypeptide,carbohydrate, nucleic acid, lipid, hapten, or fragment thereof, or othernaturally occurring or synthetic compound. In one embodiment, the targetantigen is a polypeptide.

The terms “antigen-binding fragment,” “antigen-binding domain,” and“antigen-binding region” refer to a portion of an antibody thatcomprises the amino acid residues that interact with an antigen (e.g., apolypeptide, carbohydrate, nucleic acid, lipid, hapten, or fragmentthereof, or other naturally occurring or synthetic compound) and conferon the binding agent its specificity and affinity for the antigen (e.g.,complementarity determining regions (CDRs)).

The term “specific binding,” “specifically binds to,” or “specific for”a particular polypeptide or an epitope on a particular polypeptidetarget can be exhibited, for example, by a molecule (e.g., an antibody)having a dissociation constant (K_(d)) for the target of at least about10⁻⁴ M, at least about 10⁻⁵ M, at least about 10⁻⁶ M, at least about10⁻⁷ M, at least about 10⁻⁸ M, at least about 10⁻⁹ M, at least about10⁻¹⁰ M, at least about 10⁻¹¹ M, or at least about 10⁻¹² M. In oneembodiment, the term “specific binding” refers to binding where amolecule binds to a particular polypeptide or epitope on a particularpolypeptide without substantially binding to any other polypeptide orpolypeptide epitope.

A 4-chain antibody unit is a heterotetrameric glycoprotein composed oftwo identical light (L) chains and two identical heavy (H) chains. Inthe case of IgGs, the 4-chain unit is generally about 150,000 daltons.Each L chain is linked to an H chain by one covalent disulfide bond,while the two H chains are linked to each other by one or more disulfidebonds depending on the H chain isotype. Each H and L chain also hasregularly spaced intrachain disulfide bridges. Each H chain has at theN-terminus, a variable domain (VH) followed by three constant domains(CH) for each of the α and γ chains and four CH domains for μ and εisotypes. Each L chain has at the N-terminus, a variable domain (VL)followed by a constant domain (CL) at its other end. The VL is alignedwith the VH and the CL is aligned with the first constant domain of theheavy chain (CH1). Particular amino acid residues are believed to forman interface between the light chain and heavy chain variable domains.The pairing of a VH and VL together forms a single antigen-binding site.For the structure and properties of the different classes of antibodies,see, e.g., Basic and Clinical Immunology, 8th edition, Stites et al.(eds.), Appleton & Lange, Norwalk, Conn., 1994, page 71 and Chapter 6.

The term “variable region” or “variable domain” refers to a portion ofthe light or heavy chains of an antibody that is generally located atthe amino-terminal of the light or heavy chain and has a length of about120 to 130 amino acids in the heavy chain and about 100 to 110 aminoacids in the light chain, and are used in the binding and specificity ofeach particular antibody for its particular antigen. The variable regionof the heavy chain may be referred to as “VH.” The variable region ofthe light chain may be referred to as “VL.” The term “variable” refersto the fact that certain segments of the variable regions differextensively in sequence among antibodies. The V region mediates antigenbinding and defines specificity of a particular antibody for itsparticular antigen. However, the variability is not evenly distributedacross the 110-amino acid span of the variable regions. Instead, the Vregions consist of less variable (e.g., relatively invariant) stretchescalled framework regions (FRs) of about 15-30 amino acids separated byshorter regions of greater variability (e.g., extreme variability)called “hypervariable regions” that are each about 9-12 amino acidslong. The variable regions of heavy and light chains each comprise fourFRs, largely adopting a R sheet configuration, connected by threehypervariable regions, which form loops connecting, and in some casesforming part of, the R sheet structure. The hypervariable regions ineach chain are held together in close proximity by the FRs and, with thehypervariable regions from the other chain, contribute to the formationof the antigen-binding site of antibodies (see, e.g., Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md., 1991)). Theconstant regions are not involved directly in binding an antibody to anantigen, but exhibit various effector functions, such as participationof the antibody in antibody dependent cellular cytotoxicity (ADCC) andcomplement dependent cytotoxicity (CDC). The variable regions differextensively in sequence between different antibodies. The variability insequence is concentrated in the CDRs while the less variable portions inthe variable region are referred to as framework regions (FR). The CDRsof the light and heavy chains are primarily responsible for theinteraction of the antibody with antigen. In specific embodiments, thevariable region is a human variable region.

The term “variable region residue numbering as in Kabat” or “amino acidposition numbering as in Kabat”, and variations thereof, refers to thenumbering system used for heavy chain variable regions or light chainvariable regions of the compilation of antibodies in Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991). Using thisnumbering system, the actual linear amino acid sequence may containfewer or additional amino acids corresponding to a shortening of, orinsertion into, a FR or CDR of the variable domain. For example, a heavychain variable domain may include a single amino acid insert (residue52a according to Kabat) after residue 52 of H2 and inserted residues(e.g., residues 82a, 82b, and 82c, etc., according to Kabat) after heavychain FR residue 82. The Kabat numbering of residues may be determinedfor a given antibody by alignment at regions of homology of the sequenceof the antibody with a “standard” Kabat numbered sequence. The Kabatnumbering system is generally used when referring to a residue in thevariable domain (approximately residues 1-107 of the light chain andresidues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences ofImmunological Interest. 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991)). The “EU numbering system”or “EU index” is generally used when referring to a residue in animmunoglobulin heavy chain constant region (e.g., the EU index reportedin Kabat et al., supra). The “EU index as in Kabat” refers to theresidue numbering of the human IgG 1 EU antibody. Other numberingsystems have been described, including, for example, by AbM, Chothia,Contact, IMGT and AHon.

An “intact” antibody is one comprising an antigen-binding site as wellas a CL and at least heavy chain constant regions, CH1, CH2 and CH3. Theconstant regions may include human constant regions or amino acidsequence variants thereof. Preferably, an intact antibody has one ormore effector functions.

The term “antibody fragment” refers to a portion of an intact antibody,preferably the antigen-binding or variable region of the intactantibody. Examples of antibody fragments include, without limitation,Fab, Fab′, F(ab′)2, and Fv fragments; diabodies and di-diabodies (see,e.g., Holliger et al., Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 6444-8;Lu et al., J. Biol. Chem. 2005, 280, 19665-72; Hudson et al., Nat. Med.2003, 9, 129-134; WO 93/11161; and U.S. Pat. Nos. 5,837,242 and6,492,123); single-chain antibody molecules (see, e.g., U.S. Pat. Nos.4,946,778; 5,260,203; 5,482,858 and 5,476,786); dual variable domainantibodies (see, e.g., U.S. Pat. No. 7,612,181); single variable domainantibodies (SdAbs) (see, e.g., Woolven et al., Immunogenetics 1999, 50,98-101 Streltsov et al., Proc. Natl. Acad. Sci. U.S.A. 2004, 101,12444-12449); and multispecific antibodies formed from antibodyfragments.

The term “functional fragment,” “binding fragment,” or “antigen-bindingfragment” of an antibody refers to a molecule that exhibits at least oneof the biological functions attributed to the intact antibody, thefunction comprising at least binding to the target antigen.

The term “heavy chain” when used in reference to an antibody refers to apolypeptide chain of about 50-70 kDa, wherein the amino-terminal portionincludes a variable region of about 120 to 130 or more amino acids and acarboxyl-terminal portion that includes a constant region. The constantregion can be one of five distinct types, (e.g., isotypes) referred toas alpha (α), delta (δ), epsilon (ε), gamma (γ) and mu (μ), based on theamino acid sequence of the heavy chain constant region. The distinctheavy chains differ in size: α, δ and γ contain approximately 450 aminoacids, while p and F contain approximately 550 amino acids. Whencombined with a light chain, these distinct types of heavy chains giverise to five well known classes (e.g., isotypes) of antibodies, IgA,IgD, IgE, IgG and IgM, respectively, including four subclasses of IgG,namely IgG1, IgG2, IgG3, and IgG4. A heavy chain can be a human heavychain.

The term “light chain” when used in reference to an antibody refers to apolypeptide chain of about 25 kDa, wherein the amino-terminal portionincludes a variable region of about 100 to about 110 or more amino acidsand a carboxyl-terminal portion that includes a constant region. Theapproximate length of a light chain is 211 to 217 amino acids. There aretwo distinct types, referred to as kappa (x) of lambda (k) based on theamino acid sequence of the constant domains. Light chain amino acidsequences are well known in the art. A light chain can be a human lightchain.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,e.g., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts, and each monoclonal antibody will typically recognize asingle epitope on the antigen. In specific embodiments, a “monoclonalantibody,” as used herein, is an antibody produced by a single hybridomaor other cell, wherein the antibody binds to only a beta klotho epitopeas determined, for example, by ELISA or other antigen-binding orcompetitive binding assay known in the art. The term “monoclonal” is notlimited to any particular method for making the antibody. For example,the monoclonal antibodies useful in the present disclosure may beprepared by the hybridoma methodology first described by Kohler et al.,Nature 1975, 256, 495; or may be made using recombinant DNA methods inbacterial, eukaryotic animal or plant cells (see, e.g., U.S. Pat. No.4,816,567). The “monoclonal antibodies” may also be isolated from phageantibody libraries using the techniques described in Clackson et al.,Nature 1991, 352, 624-628 and Marks et al., J. Mol. Biol. 1991, 222,581-597, for example. Other methods for the preparation of clonal celllines and of monoclonal antibodies expressed thereby are well known inthe art (see, for example, Chapter 11 in: Short Protocols in MolecularBiology, (2002) 5th Ed., Ausubel et al., eds., John Wiley and Sons, NewYork). Exemplary methods of producing monoclonal antibodies are providedin the Examples herein.

“Humanized” forms of nonhuman (e.g., murine) antibodies are chimericantibodies that include human immunoglobulins (e.g., recipient antibody)in which the native CDR residues are replaced by residues from thecorresponding CDR of a nonhuman species (e.g., donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, one or more FR regionresidues of the human immunoglobulin are replaced by correspondingnonhuman residues. Furthermore, humanized antibodies can compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. A humanized antibody heavy or light chain can comprisesubstantially all of at least one or more variable regions, in which allor substantially all of the CDRs correspond to those of a nonhumanimmunoglobulin and all or substantially all of the FRs are those of ahuman immunoglobulin sequence. In certain embodiments, the humanizedantibody will comprise at least a portion of an immunoglobulin constantregion (Fc), typically that of a human immunoglobulin. For furtherdetails, see, Jones et al., Nature 1986, 321, 522-525; Riechmann et al.,Nature 1988, 332, 323-329; Presta, Curr. Opin. Biotechnol. 1992, 3,394-398; Carter et al., Proc. Natl. Acad. Sci. U.S.A. 1992, 89,4285-4289; and U.S. Pat. Nos. 6,800,738, 6,719,971, 6,639,055,6,407,213, and 6,054,297.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen-bindingresidues. Human antibodies can be produced using various techniquesknown in the art, including phage-display libraries (Hoogenboom andWinter, J. Mol. Biol. 1991, 227, 381; Marks et al., J. Mol. Biol. 1991,222, 581) and yeast display libraries (Chao et al., Nature Protocols2006, 1, 755-768). Also available for the preparation of humanmonoclonal antibodies are methods described in Cole et al., MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner etal., J. Immunol. 1991, 147, 86-95. See also van Dijk and van de Winkel,Curr. Opin. Pharmacol. 2001, 5, 368-374. Human antibodies can beprepared by administering the antigen to a transgenic animal that hasbeen modified to produce such antibodies in response to antigenicchallenge, but whose endogenous loci have been disabled, e.g., mice(see, e.g., Jakobovits, Curr. Opin. Biotechnol. 1995, 6, 561-566;Brüggemann and Taussing, Curr. Opin. Biotechnol. 1997, 8, 455-458; andU.S. Pat. Nos. 6,075,181 and 6,150,584 regarding XENOMOUSE™ technology).See also, for example, Li et al., Proc. Natl. Acad. Sci. U.S.A. 2006,103, 3557-3562 regarding human antibodies generated via a human B-cellhybridoma technology.

A “CDR” refers to one of three hypervariable regions (H1, H2, or H3)within the non-framework region of the immunoglobulin (Ig or antibody)VH j-sheet framework, or one of three hypervariable regions (L1, L2, orL3) within the non-framework region of the antibody VL j-sheetframework. Accordingly, CDRs are variable region sequences interspersedwithin the framework region sequences. CDR regions are well known tothose skilled in the art and have been defined by, for example, Kabat asthe regions of most hypervariability within the antibody variable (V)domains. Kabat et al., J. Biol. Chem. 1977, 252, 6609-6616; Kabat, Adv.Protein Chem. 1978, 32, 1-75. CDR region sequences also have beendefined structurally by Chothia as those residues that are not part ofthe conserved β-sheet framework, and thus are able to adapt differentconformations. Chothia and Lesk, J. Mol. Biol. 1987, 196, 901-917. Bothterminologies are well recognized in the art. CDR region sequences havealso been defined by AbM, Contact and IMGT. The positions of CDRs withina canonical antibody variable region have been determined by comparisonof numerous structures. Al-Lazikani et al., J. Mol. Biol. 1997, 273,927-948; Morea et al., Methods. 2000, 20, 267-279. Because the number ofresidues within a hypervariable region varies in different antibodies,additional residues relative to the canonical positions areconventionally numbered with a, b, c and so forth next to the residuenumber in the canonical variable region numbering scheme. Al-Lazikani etal., supra (1997). Such nomenclature is similarly well known to thoseskilled in the art.

The term “hypervariable region”, “HVR”, or “HF”, when used herein refersto the regions of an antibody variable region that are hypervariable insequence and/or form structurally defined loops. Generally, antibodiescomprise six hypervariable regions; three in the VH (H1, H2, H3), andthree in the VL (L1, L2, L3). A number of hypervariable regiondelineations are in use and are encompassed herein. The KabatComplementarity Determining Regions (CDRs) are based on sequencevariability and are the most commonly used (see, e.g., Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991)). Chothiarefers instead to the location of the structural loops. See, e.g.,Chothia and Lesk, J. Mol. Biol. 1987, 196, 901-917. The end of theChothia CDR-H1 loop when numbered using the Kabat numbering conventionvaries between H32 and H34 depending on the length of the loop (this isbecause the Kabat numbering scheme places the insertions at H35A andH35B; if neither 35A nor 35B is present, the loop ends at 32; if only35A is present, the loop ends at 33; if both 35A and 35B are present,the loop ends at 34). The AbM hypervariable regions represent acompromise between the Kabat CDRs and Chothia structural loops, and areused by Oxford Molecular's AbM antibody modeling software (see, e.g.,Martin, in Antibody Engineering, Vol. 2, Chapter 3, Springer Verlag).The “contact” hypervariable regions are based on an analysis of theavailable complex crystal structures. The residues from each of thesehypervariable regions or CDRs are noted below.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain, including, for example, native sequence Fcregions, recombinant Fc regions, and variant Fc regions. Although theboundaries of the Fc region of an immunoglobulin heavy chain might vary,the human IgG heavy chain Fc region is often defined to stretch from anamino acid residue at position Cys226, or from Pro230, to thecarboxyl-terminus thereof. The C-terminal lysine (residue 447 accordingto the EU numbering system) of the Fc region may be removed, forexample, during production or purification of the antibody, or byrecombinantly engineering the nucleic acid encoding a heavy chain of theantibody. Accordingly, a composition of intact antibodies may compriseantibody populations with all K447 residues removed, antibodypopulations with no K447 residues removed, and antibody populationshaving a mixture of antibodies with and without the K447 residue.

“Cycloalkyl” indicates a non-aromatic, fully saturated carbocyclic ringhaving the indicated number of carbon atoms, for example, 3 to 10, or 3to 8, or 3 to 6 ring carbon atoms. Cycloalkyl groups may be monocyclicor polycyclic (e.g., bicyclic, tricyclic). Examples of cycloalkyl groupsinclude cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl, as well asbridged and caged ring groups (e.g., norbornane, bicyclo[2.2.2]octane).In addition, one ring of a polycyclic cycloalkyl group may be aromatic,provided the polycyclic cycloalkyl group is bound to the parentstructure via a non-aromatic carbon. For example, a1,2,3,4-tetrahydronaphthalen-1-yi group (wherein the moiety is bound tothe parent structure via a non-aromatic carbon atom) is a cycloalkylgroup, while 1,2,3,4-tetrahydronaphthalen-5-yl (wherein the moiety isbound to the parent structure via an aromatic carbon atom) is notconsidered a cycloalkyl group. Examples of polycyclic cycloalkyl groupsconsisting of a cycloalkyl group fused to an aromatic ring are describedbelow.

“Cycloalkenyl” indicates a non-aromatic carbocyclic ring, containing theindicated number of carbon atoms (e.g., 3 to 10, or 3 to 8, or 3 to 6ring carbon atoms) and at least one carbon-carbon double bond.Cycloalkenyl groups may be monocyclic or polycyclic (e.g., bicyclic,tricyclic). Examples of cycloalkenyl groups include cyclopropenyl,cyclobutenyl, cyclopentenyl, cyclopentadienyl, and cyclohexenyl, as wellas bridged and caged ring groups (e.g., bicyclo[2.2.2]octene). Inaddition, one ring of a polycyclic cycloalkenyl group may be aromatic,provided the polycyclic alkenyl group is bound to the parent structurevia a non-aromatic carbon atom. For example, inden-1-yl (wherein themoiety is bound to the parent structure via a non-aromatic carbon atom)is considered a cycloalkenyl group, while inden-4-yl (wherein the moietyis bound to the parent structure via an aromatic carbon atom) is notconsidered a cycloalkenyl group. Examples of polycyclic cycloalkenylgroups consisting of a cycloalkenyl group fused to an aromatic ring aredescribed below.

“Cycloalkynyl” refers to an unsaturated hydrocarbon group within acycloalkyl having at least one site of acetylenic unsaturation (i.e.,having at least one moiety of the formula C≡C). Cycloalkynyl can consistof one ring, such as cyclooctyne, or multiple rings. One cycloalkynylmoiety is an unsaturated cyclic hydrocarbon having from 5 to 10 annularcarbon atoms (a “C₅-C₁₀ cycloalkynyl”). Examples include cyclopentyne,cyclohexyne, cycloheptyne, cyclooctyne, cyclononyne, and the like.

“Aryl” indicates an aromatic carbocyclic ring having the indicatednumber of carbon atoms, for example, 6 to 12 or 6 to 10 carbon atoms.Aryl groups may be monocyclic or polycyclic (e.g., bicyclic, tricyclic).In some instances, both rings of a polycyclic aryl group are aromatic(e.g., naphthyl). In other instances, polycyclic aryl groups may includea non-aromatic ring fused to an aromatic ring, provided the polycyclicaryl group is bound to the parent structure via an atom in the aromaticring. Thus, a 1,2,3,4-tetrahydronaphthalen-5-yl group (wherein themoiety is bound to the parent structure via an aromatic carbon atom) isconsidered an aryl group, while 1,2,3,4-tetrahydronaphthalen-1-yl(wherein the moiety is bound to the parent structure via a non-aromaticcarbon atom) is not considered an aryl group. Similarly, a1,2,3,4-tetrahydroquinolin-8-yl group (wherein the moiety is bound tothe parent structure via an aromatic carbon atom) is considered an arylgroup, while 1,2,3,4-tetrahydroquinolin-1-yl group (wherein the moietyis bound to the parent structure via a non-aromatic nitrogen atom) isnot considered an aryl group. However, the term “aryl” does notencompass or overlap with “heteroaryl”, as defined herein, regardless ofthe point of attachment (e.g., both quinolin-5-yl and quinolin-2-yl areheteroaryl groups). In some instances, aryl is phenyl or naphthyl. Incertain instances, aryl is phenyl. Additional examples of aryl groupscomprising an aromatic carbon ring fused to a non-aromatic ring aredescribed below.

The term “DAR” refers to a drug-antibody ratio of anoligonucleotide-antibody conjugate, more specifically animmunomodulating polynucleotide-antibody ratio. In some instances, forexample, an oligonucleotide-antibody conjugate may be described hereinas having a DAR of 1 or as a DAR1 conjugate, wherein theoligonucleotide-antibody ratio is 1-to-1. In other instances, an anoligonucleotide-antibody conjugate may be described herein as having aDAR of 2 or as a DAR2 conjugate, wherein the oligonucleotide-antibodyratio is 2-to-1.

“Heteroaryl” indicates an aromatic ring containing the indicated numberof atoms (e.g., 5 to 12, or 5 to 10 membered heteroaryl) made up of oneor more heteroatoms (e.g., 1, 2, 3 or 4 heteroatoms) selected from N, Oand S and with the remaining ring atoms being carbon. Heteroaryl groupsdo not contain adjacent S and O atoms. In some embodiments, the totalnumber of S and O atoms in the heteroaryl group is not more than 2. Insome embodiments, the total number of S and O atoms in the heteroarylgroup is not more than 1. Unless otherwise indicated, heteroaryl groupsmay be bound to the parent structure by a carbon or nitrogen atom, asvalency permits. For example, “pyridyl” includes 2-pyridyl, 3-pyridyland 4-pyridyl groups, and “pyrrolyl” includes 1-pyrrolyl, 2-pyrrolyl and3-pyrrolyl groups.

In some instances, a heteroaryl group is monocyclic. Examples includepyrrole, pyrazole, imidazole, triazole (e.g., 1,2,3-triazole,1,2,4-triazole, 1,2,4-triazole), tetrazole, furan, isoxazole, oxazole,oxadiazole (e.g., 1,2,3-oxadiazole, 1,2,4-oxadiazole, 1,3,4-oxadiazole),thiophene, isothiazole, thiazole, thiadiazole (e.g., 1,2,3-thiadiazole,1,2,4-thiadiazole, 1,3,4-thiadiazole), pyridine, pyridazine, pyrimidine,pyrazine, triazine (e.g., 1,2,4-triazine, 1,3,5-triazine) and tetrazine.

In some instances, both rings of a polycyclic heteroaryl group arearomatic. Examples include indole, isoindole, indazole, benzoimidazole,benzotriazole, benzofuran, benzoxazole, benzoisoxazole, benzoxadiazole,benzothiophene, benzothiazole, benzoisothiazole, benzothiadiazole,1H-pyrrolo[2,3-b]pyridine, 1H-pyrazolo[3,4-b]pyridine,3H-imidazo[4,5-b]pyridine, 3H-[1,2,3]triazolo[4,5-b]pyridine,1H-pyrrolo[3,2-b]pyridine, 1H-pyrazolo[4,3-b]pyridine,1H-imidazo[4,5-b]pyridine, 1H-[1,2,3]triazolo[4,5-b]pyridine,1H-pyrrolo[2,3-c]pyridine, 1H-pyrazolo[3,4-c]pyridine,3H-imidazo[4,5-c]pyridine, 3H-[1,2,3]triazolo[4,5-c]pyridine,1H-pyrrolo[3,2-c]pyridine, 1H-pyrazolo[4,3-c]pyridine,1H-imidazo[4,5-c]pyridine, 1H-[1,2,3]triazolo[4,5-c]pyridine,furo[2,3-b]pyridine, oxazolo[5,4-b]pyridine, isoxazolo[5,4-b]pyridine,[1,2,3]oxadiazolo[5,4-b]pyridine, furo[3,2-b]pyridine,oxazolo[4,5-b]pyridine, isoxazolo[4,5-b]pyridine,[1,2,3]oxadiazolo[4,5-b]pyridine, furo[2,3-c]pyridine,oxazolo[5,4-c]pyridine, isoxazolo[5,4-c]pyridine,[1,2,3]oxadiazolo[5,4-c]pyridine, furo[3,2-c]pyridine,oxazolo[4,5-c]pyridine, isoxazolo[4,5-c]pyridine,[1,2,3]oxadiazolo[4,5-c]pyridine, thieno[2,3-b]pyridine,thiazolo[5,4-b]pyridine, isothiazolo[5,4-b]pyridine,[1,2,3]thiadiazolo[5,4-b]pyridine, thieno[3,2-b]pyridine,thiazolo[4,5-b]pyridine, isothiazolo[4,5-b]pyridine,[1,2,3]thiadiazolo[4,5-b]pyridine, thieno[2,3-c]pyridine,thiazolo[5,4-c]pyridine, isothiazolo[5,4-c]pyridine,[1,2,3]thiadiazolo[5,4-c]pyridine, thieno[3,2-c]pyridine,thiazolo[4,5-c]pyridine, isothiazolo[4,5-c]pyridine,[1,2,3]thiadiazolo[4,5-c]pyridine, quinoline, isoquinoline, cinnoline,quinazoline, quinoxaline, phthalazine, naphthyridine (e.g.,1,8-naphthyridine, 1,7-naphthyridine, 1,6-naphthyridine,1,5-naphthyridine, 2,7-naphthyridine, 2,6-naphthyridine),imidazo[1,2-a]pyridine, 1H-pyrazolo[3,4-d]thiazole,1H-pyrazolo[4,3-d]thiazole and imidazo[2,1-b]thiazole.

In other instances, polycyclic heteroaryl groups may include anon-aromatic ring (e.g., cycloalkyl, cycloalkenyl, heterocycloalkyl,heterocycloalkenyl) fused to a heteroaryl ring, provided the polycyclicheteroaryl group is bound to the parent structure via an atom in thearomatic ring. For example, a 4,5,6,7-tetrahydrobenzo[d]thiazol-2-ylgroup (wherein the moiety is bound to the parent structure via anaromatic carbon atom) is considered a heteroaryl group, while4,5,6,7-tetrahydrobenzo[d]thiazol-5-yl (wherein the moiety is bound tothe parent structure via a non-aromatic carbon atom) is not considered aheteroaryl group. Examples of polycyclic heteroaryl groups consisting ofa heteroaryl ring fused to a non-aromatic ring are described below.

As used herein, the terms “including,” “containing,” and “comprising”are used in their open, non-limiting sense. It is also understood thataspects and embodiments of the invention described herein may include“consisting” and/or “consisting essentially of” aspects and embodiments.

It is understood that, whether the term “about” is used explicitly ornot, every quantity given herein is meant to refer to the actual givenvalue, and it is also meant to refer to the approximation to such givenvalue that would reasonably be inferred based on the ordinary skill inthe art, including equivalents and approximations due to theexperimental and/or measurement conditions for such given value.

As used herein, a “carrier” includes pharmaceutically acceptablecarriers, excipients, or stabilizers that are nontoxic to the cell ormammal being exposed thereto at the dosages and concentrations employed.Often the physiologically acceptable carrier is an aqueous pH bufferedsolution. Non-limiting examples of physiologically acceptable carriersinclude buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

As used herein, the term “effective amount” or “therapeuticallyeffective amount” of a substance is at least the minimum concentrationrequired to bring about a measurable improvement or prevention of aparticular disorder. An effective amount herein may vary according tofactors such as the disease state, age, sex, and weight of the patient,and the ability of the substance to elicit a desired response in theindividual. An effective amount is also one in which any toxic ordetrimental effects of the treatment are outweighed by thetherapeutically beneficial effects. In reference to cancer, an effectiveamount comprises an amount sufficient to cause a tumor to shrink and/orto decrease the growth rate of the tumor (such as to suppress tumorgrowth) or to prevent or delay other unwanted cell proliferation incancer. In some embodiments, an effective amount is an amount sufficientto delay development of cancer. In some embodiments, an effective amountis an amount sufficient to prevent or delay recurrence. In someembodiments, an effective amount is an amount sufficient to reducerecurrence rate in the individual. An effective amount can beadministered in one or more administrations. The effective amount of thedrug or composition may: (i) reduce the number of cancer cells; (ii)reduce tumor size; (iii) inhibit, retard, slow to some extent andpreferably stop cancer cell infiltration into peripheral organs; (iv)inhibit (i.e., slow to some extent and preferably stop) tumormetastasis; (v) inhibit tumor growth; (vi) prevent or delay occurrenceand/or recurrence of tumor; (vii) reduce recurrence rate of tumor,and/or (viii) relieve to some extent one or more of the symptomsassociated with the cancer. An effective amount can be administered inone or more administrations. For purposes of this disclosure, aneffective amount of drug, compound, or pharmaceutical composition is anamount sufficient to accomplish prophylactic or therapeutic treatmenteither directly or indirectly. As is understood in the clinical context,an effective amount of a drug, compound, or pharmaceutical compositionmay or may not be achieved in conjunction with another drug, compound,or pharmaceutical composition. Thus, an “effective amount” may beconsidered in the context of administering one or more therapeuticagents, and a single agent may be considered to be given in an effectiveamount if, in conjunction with one or more other agents, a desirableresult may be or is achieved.

A “package insert” refers to instructions customarily included incommercial packages of medicaments that contain information about theindications customarily included in commercial packages of medicamentsthat contain information about the indications, usage, dosage,administration, contraindications, other medicaments to be combined withthe packaged product, and/or warnings concerning the use of suchmedicaments, etc.

The terms “protein,” “polypeptide” and “peptide” are used herein torefer to polymers of amino acids of any length. The polymer may belinear or branched, it may comprise modified amino acids, and it may beinterrupted by non-amino acids. The terms also encompass an amino acidpolymer that has been modified naturally or by intervention; forexample, disulfide bond formation, glycosylation, lipidation,acetylation, phosphorylation, or any other manipulation or modification,such as conjugation with a labeling component. Typically, a protein foruse herein will have a molecular weight of at least about 5-20 kDa,alternatively at least about 20-100 kDa, or at least about 100 kDa. Alsoincluded within the definition are, for example, proteins containing oneor more analogs of an amino acid (including, for example, unnaturalamino acids, etc.), as well as other modifications known in the art.

A “pharmaceutically acceptable salt” is a salt form that is non-toxic,biologically tolerable, or otherwise biologically suitable foradministration to the subject. See generally Berge et al. (1977) J.Pharm. Sci. 66, 1. Particular pharmaceutically acceptable salts arethose that are pharmacologically effective and suitable for contact withthe tissues of subjects without undue toxicity, irritation, or allergicresponse. Pharmaceutically acceptable salts include, without limitation,acid addition salts, formed with inorganic acids such as hydrochloricacid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, andthe like; or formed with organic acids such as acetic acid, oxalic acid,propionic acid, succinic acid, maleic acid, tartaric acid and the like.These salts may be derived from inorganic or organic acids. Non-limitingexamples of pharmaceutically acceptable salts include sulfates,pyrosulfates, bisulfates, sulfites, bisulfites, phosphates,monohydrogen-phosphates, dihydrogenphosphates, metaphosphates,pyrophosphates, chlorides, bromides, iodides, acetates, propionates,decanoates, caprylates, acrylates, formates, isobutyrates, caproates,heptanoates, propiolates, oxalates, malonates, succinates, suberates,sebacates, fumarates, maleates, butyne-1,4-dioates, hexyne-1,6-dioates,benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates,hydroxybenzoates, methoxybenzoates, phthalates, sulfonates,methylsulfonates, propylsulfonates, besylates, xylenesulfonates,naphthalene-1-sulfonates, naphthalene-2-sulfonates, phenylacetates,phenylpropionates, phenylbutyrates, citrates, lactates,γ-hydroxybutyrates, glycolates, tartrates, and mandelates. In someembodiments, pharmaceutically acceptable salts are formed when an acidicproton present in the parent compound either is replaced by a metal ion,e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; orcoordinates with an organic base. Salts derived from pharmaceuticallyacceptable organic non-toxic bases include, without limitation, salts ofprimary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines and basic ionexchange resins, such as isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol,tromethamine, trimetharnine, dicyclohexylamine, caffeine, procaine,hydrabamine, choline, betaine, ethylenediamine, glucosamine,N-ethylglucamine, N-methylglucamine, theobromine, purines, piperazine,piperidine, N-ethylpiperidine, polyamine resins, amino acids such aslysine, arginine, histidine, and the like. Examples of pharmaceuticallyacceptable base addition salts include those derived from inorganicbases such as sodium, potassium, lithium, ammonium, calcium, magnesium,iron, zinc, copper, manganese, aluminum salts and the like. In someembodiments, the organic non-toxic bases are L-amino acids, such asL-lysine and L-arginine, tromethamine, N-ethylglucamine andN-methylglucamine. Acceptable inorganic bases include, withoutlimitation, aluminum hydroxide, calcium hydroxide, potassium hydroxide,sodium carbonate, sodium hydroxide, and the like. Lists of othersuitable pharmaceutically acceptable salts are found in Remington'sPharmaceutical Sciences, 17th Edition, Mack Publishing Company, Easton,Pa., 1985.

A “solvate” is formed by the interaction of a solvent and a compound.Suitable solvents include, for example, water and alcohols (e.g.,ethanol). Solvates include hydrates having any ratio of compound towater, such as monohydrates, dihydrates and hemi-hydrates.

A “subject,” “patient” or “individual” includes a mammal, such as ahuman or other animal, and typically is human. In some embodiments, thesubject, e.g., patient, to whom the therapeutic agents and compositionsare administered, is a mammal, typically a primate, such as a human. Insome embodiments, the primate is a monkey or an ape. The subject can bemale or female and can be any suitable age, including infant, juvenile,adolescent, adult, and geriatric subjects. In some embodiments, thesubject is a non-primate mammal, such as a rodent, a dog, a cat, a farmanimal, such as a cow or a horse, etc.

The term “cancer” or “tumor” refers to the presence of cells possessingcharacteristics typical of cancer-causing cells, such as uncontrolledproliferation, immortality, metastatic potential, rapid growth andproliferation rate, and certain characteristic morphological features.Cancer cells are often in the form of a solid tumor, which is detectableon the basis of tumor mass, e.g., by procedures such as CAT scan, MRimaging, X-ray, ultrasound or palpation, and/or which is detectablebecause of the expression of one or more cancer-specific antigens in asample obtainable from a patient. In some embodiments, a solid tumordoes not need to have measurable dimensions. Cancer cells may also inthe form of a liquid tumor, which cancer cells may exist alone ordisseminated within an animal. As used herein, the terms “disseminatedtumor” and “liquid tumor” are used interchangeably, and include, withoutlimitation, leukemia and lymphoma and other blood cell cancers.

The term “leukemia” refers to a type of cancer of the blood or bonemarrow characterized by an abnormal increase of immature white bloodcells called “blasts.” Leukemia is a broad term covering a spectrum ofdiseases. In turn, it is part of the even broader group of diseasesaffecting the blood, bone marrow, and lymphoid system, which are allknown as hematological neoplasms. Leukemias can be divided into fourmajor classifications, acute lymphocytic (or lymphoblastic) leukemia(ALL), acute myelogenous (or myeloid or non-lymphatic) leukemia (AML),chronic lymphocytic leukemia (CLL), and chronic myelogenous leukemia(CMIL). Further types of leukemia include Hairy cell leukemia (HCL),T-cell prolymphocytic leukemia (T-PLL), large granular lymphocyticleukemia, and adult T-cell leukemia.

The term “lymphoma” refers to a group of blood cell tumors that developfrom lymphatic cells. The two main categories of lymphomas are Hodgkinlymphomas (HL) and non-Hodgkin lymphomas (NHL) Lymphomas include anyneoplasms of the lymphatic tissues. The main classes are cancers of thelymphocytes, a type of white blood cell that belongs to both the lymphand the blood and pervades both.

As used herein, the term “cancer” includes premalignant as well asmalignant cancers, and also includes primary tumors (e.g., those whosecells have not migrated to sites in the subject's body other than thesite of the original tumor) and secondary tumors (e.g., those arisingfrom metastasis, the migration of tumor cells to secondary sites thatare different from the site of the original tumor), recurrent cancer andrefractory cancer.

The terms “cancer recurrence” and “cancer relapse” are usedinterchangeably and refer to the return of a sign, symptom or diseaseafter a remission. The recurrent cancer cells may re-appear in the samesite of the primary tumor or in another location, such as in secondarycancer. The cancer cells may re-appear in the same diseased form as theprimary cancer or a different diseased form. For example, in someembodiments, a primary cancer is a solid tumor, and the recurrent canceris a liquid tumor. In other embodiments, a primary cancer is a liquidtumor, and the recurrent cancer is a solid tumor. In yet otherembodiments, the primary cancer and the recurrent cancer are both solidtumors, or both liquid tumors. In some embodiments, the recurrent tumorexpresses at least one tumor-associated antigen that is also expressedby the primary tumor.

The term “refractory cancer” as used herein refers to a cancer that doesnot respond to a treatment, for example, a cancer that is resistant atthe beginning of treatment (e.g., treatment with an immunotherapy) or acancer that may become resistant during treatment. The terms “respond,”“response” or “responsiveness” refer to an anti-cancer response, e.g. inthe sense of reduction of tumor size or inhibiting tumor growth. Theterms can also refer to an improved prognosis, for example, as reflectedby an increased time to recurrence, which is the period to firstrecurrence censoring for second primary cancer as a first event or deathwithout evidence of recurrence, or an increased overall survival, whichis the period from treatment to death from any cause. To respond or tohave a response means there is a beneficial endpoint attained whenexposed to a stimulus. Alternatively, a negative or detrimental symptomis minimized, mitigated or attenuated on exposure to a stimulus. It willbe appreciated that evaluating the likelihood that a tumor or subjectwill exhibit a favorable response is equivalent to evaluating thelikelihood that the tumor or subject will not exhibit favorable response(i.e., will exhibit a lack of response or be non-responsive).

As used herein, cancers include, but are not limited to, melanomas,breast cancer, lung cancer, bronchus cancer, colorectal cancer, prostatecancer, pancreatic cancer, stomach cancer, ovarian cancer, urinarybladder cancer, brain or central nervous system cancer, peripheralnervous system cancer, esophageal cancer, cervical cancer, uterine orendometrial cancer, cancer of the oral cavity or pharynx, liver cancer,kidney cancer, testicular cancer, biliary tract cancer, small bowel orappendix cancer, salivary gland cancer, thyroid gland cancer, adrenalgland cancer, osteosarcoma, chondrosarcoma, cancer of hematologictissues, B cell cancer, e.g., multiple myeloma, Waldenström'smacroglobulinemia, the heavy chain diseases, such as, for example, alphachain disease, gamma chain disease, and mu chain disease, benignmonoclonal qammopathy, and immunocytic amyloidosis, and the like. Othernon-limiting examples of types of cancers applicable to the methodsencompassed by the present invention include human sarcomas andcarcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, colorectal cancer, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, liver cancer,choriocarcinoma, sominoma, embryonal carcinoma, Wilms' tumor, cervicalcancer, bone cancer, brain tumor, testicular cancer, lung carcinoma,small cell lung carcinoma, bladder carcinoma, epithelial carcinoma,glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma,pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma,meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias, e.g.,acute lymphocytic leukemia and acute myelocytic leukemia (myeloblastic,promyelocytic, myelomonocytic, monocytic and erythroleukemia); chronicleukemia (chronic myelocytic (granulocytic) leukemia and chroniclymphocytic leukemia); and polycythemia vera, lymphoma (Hodgkin'sdisease and non-Hodgkin's disease), multiple myeloma, Waldenstrom'smacroglobulinemia, and heavy chain disease. In some embodiments, cancersare epithlelial in nature and include but are not limited to, bladdercancer, breast cancer, cervical cancer, colon cancer, gynecologiccancers, renal cancer, laryngeal cancer, lung cancer, oral cancer, headand neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, orskin cancer. In other embodiments, the cancer is breast cancer, prostatecancer, lung cancer, or colon cancer. In still other embodiments, theepithelial cancer is non-small-cell lung cancer, nonpapillary renal cellcarcinoma, cervical carcinoma, ovarian carcinoma (e.g., serous ovariancarcinoma), or breast carcinoma. The epithelial cancers may becharacterized in various other ways including, but not limited to,serous, endometrioid, mucinous, clear cell, Brenner, orundifferentiated.

The term “cancer therapy” or “cancer therapeutic agent” as used herein,refers to those therapies or agents that can exert anti-tumor effect orhave an anti-tumor activity. Such anti-tumor effect or anti-tumoractivity can be exhibited as a reduction in the rate of tumor cellproliferation, viability, or metastatic activity. A possible way ofshowing anti-tumor activity is to show a decline in growth rate ofabnormal cells that arises during therapy or tumor size stability orreduction. Such activity can be assessed using accepted in vitro or invivo tumor models, including but not limited to xenograft models,allograft models, MMTV models, and other known models known in the artto investigate anti-tumor activity.

The terms “treat,” “treating,” and “treatment” are meant to includealleviating or abrogating a condition, disorder, or disease, or one ormore of the symptoms associated with the condition, disorder, ordisease; or alleviating or eradicating the cause(s) of the condition,disorder, or disease itself.

The terms “prevent,” “preventing,” and “prevention” are meant to includea method of delaying and/or precluding the onset of a condition,disorder, or disease, and/or its attendant symptoms; barring a subjectfrom acquiring a condition, disorder, or disease; or reducing asubject's risk of acquiring a condition, disorder, or disease.

The term “substituted” means that the specified group or moiety bearsone or more substituents including, but not limited to, substituentssuch as alkoxy, acyl, acyloxy, alkoxycarbonyl, carbonylalkoxy,acylamino, amino, aminoacyl, aminocarbonylamino, aminocarbonyloxy,cycloalkyl, cycloalkenyl, aryl, heteroaryl, aryloxy, cyano, azido, halo,hydroxyl, nitro, carboxyl, thiol, thioalkyl, alkyl, alkenyl, alkynyl,heterocyclyl, aralkyl, aminosulfonyl, sulfonylamino, sulfonyl, oxo, andthe like. The term “unsubstituted” means that the specified group bearsno substituents. Where the term “substituted” is used to describe astructural system, the substitution is meant to occur at anyvalency-allowed position on the system. When a group or moiety bearsmore than one substituent, it is understood that the substituents may bethe same or different from one another. In some embodiments, asubstituted group or moiety bears from one to five substituents. In someembodiments, a substituted group or moiety bears one substituent. Insome embodiments, a substituted group or moiety bears two substituents.In some embodiments, a substituted group or moiety bears threesubstituents. In some embodiments, a substituted group or moiety bearsfour substituents. In some embodiments, a substituted group or moietybears five substituents.

By “optional” or “optionally” is meant that the subsequently describedevent or circumstance may or may not occur, and that the descriptionincludes instances where the event or circumstance occurs and instancesin which it does not. For example, “optionally substituted alkyl”encompasses both “alkyl” and “substituted alkyl” as defined herein. Itwill be understood by those skilled in the art, with respect to anygroup containing one or more substituents, that such groups are notintended to introduce any substitution or substitution patterns that aresterically impractical, synthetically non-feasible, and/or inherentlyunstable. It will also be understood that where a group or moiety isoptionally substituted, the disclosure includes both embodiments inwhich the group or moiety is substituted and embodiments in which thegroup or moiety is unsubstituted.

The term “Q-tag,” as used herein, refers to a portion of a polypeptidecontaining glutamine residue that, upon transglutaminase-mediatedreaction with a compound containing —NH₂ amine, provides a conjugatecontaining the portion of polypeptide, in which the glutamine residueincludes a side chain modified to include the amide bonded to thecompound. Q-tags are known in the art. Non-limiting examples of Q-tagsare LLQGG (SEQ ID NO:172) and GGGLLQGG (SEQ ID NO:173). In someembodiments, the Q tag is attached to the C terminal of the heavy chainof the antibody. In some embodiments, the Q tag is attached to the lightchain of the antibody. In some embodiments, the Q tag is naturallyoccurring. For example, mutation of N297 to N297A exposes Q295 of theantibody, where the conjugation could occur (numbering according to EUindex, e.g., as listed in Edelman, G. M. et al., Proc. Natl. Acad. USA,63, 78-85 (1969) and Kabat, E. A. et al., Sequences of proteins ofimmunological interest. 5th Edition—US Department of Health and HumanServices, NIH publication no 91-3242, pp 662, 680, 689 (1991)). In someembodiments, the Q tag is within the Fc domain of the antibody.

II. Conjugates

Immunostimulating polynucleotides have been used in a variety oftherapeutic applications. To improve targeting specificity and in vivodistribution, the immunomodulating polynucleotides (e.g., CpG ODNs) canbe conjugated to a targeting moiety (e.g., polypeptides). Particularly,transglutaminase-mediated reaction can be used to conduct such aconjugation reaction due to its high reaction rates and suitable sitespecificity. The present disclosure provides oligonucleotide-polypeptideconjugates exhibiting favorable activity. In some embodiments, thepolypeptide is an antibody, such as an antibody heavy or light chain.

Provided herein is an oligonucleotide-antibody conjugate wherein theoligonucleotide and antibody are attached together via a linking moiety.In some embodiments, one antibody can be conjugated to one or moreoligonucleotides. In some embodiments, the oligonucleotide-antibodyconjugate is a conjugate comprising an antibody or antigen-bindingfragment thereof and one or more immunomodulating oligonucleotides (P),wherein the antibody or antigen-binding fragment is linked to one ormore Q-tag peptides (Q) comprising at least one glutamine residue,wherein each immunomodulating oligonucleotide is linked to a Q-tagpeptide via an amide bond with the glutamine residue of the Q-tagpeptide and a linker (L) as shown in Formula (A):

or a stereoisomer, a mixture of two or more diastereomers, a tautomer,or a mixture of two or more tautomers thereof; or a pharmaceuticallyacceptable salt, solvate, or hydrate thereof;wherein:

-   -   indicates the point of attachment of each Q to the antibody or        antigen-binding fragment thereof (Ab);    -   each Q is independently a Q-tag peptide sequence comprising at        least one glutamine residue;    -   each L is independently a bond or a linker moiety connected to Q        via an amide bond with the glutamine residue; and    -   each P is independently an immunomodulating oligonucleotide.

In some embodiments, the conjugate is a conjugate comprising an antibodyor antigen-binding fragment thereof and one or more immunomodulatingoligonucleotides (P), wherein the antibody or antigen-binding fragmentis linked to one or more Q-tag peptides (Q) that comprise the amino acidsequence RPQGF (SEQ ID NO:47), wherein each immunomodulatingoligonucleotide is linked to a Q-tag peptide via an amide bond with theglutamine residue of the Q-tag peptide and a linker (L) as shown inFormula (A),

or a stereoisomer, a mixture of two or more diastereomers, a tautomer,or a mixture of two or more tautomers thereof; or a pharmaceuticallyacceptable salt, solvate, or hydrate thereof;wherein:

indicates the point of attachment of each Q to the antibody orantigen-binding fragment thereof (Ab);

each Q independently comprises a Q-tag peptide comprising a peptidesequence RPQGF (SEQ ID NO:47);

each L is independently a bond or a linker moiety connected to Q via anamide bond with the glutamine residue; and

each P is independently an immunomodulating oligonucleotide.

In other embodiments, the conjugate is a conjugate comprising anantibody or antigen-binding fragment thereof and one or moreimmunomodulating oligonucleotides (P), wherein the antibody orantigen-binding fragment is linked to one or more Q-tag peptides (Q)comprising at least one glutamine residue, wherein each immunomodulatingoligonucleotide is linked to a Q-tag peptide via an amide bond with theglutamine residue of the Q-tag peptide and a linker (L) as shown informula (A),

or a stereoisomer, a mixture of two or more diastereomers, a tautomer,or a mixture of two or more tautomers thereof; or a pharmaceuticallyacceptable salt, solvate, or hydrate thereof;wherein:

indicates the point of attachment of each Q to the antibody orantigen-binding fragment thereof (Ab);

each Q is independently a Q-tag peptide comprising at least oneglutamine residue;

each L is independently a bond or a linker moiety connected to Q via anamide bond with the glutamine residue; and

each P is independently an immunomodulating oligonucleotide selectedfrom the group consisting of the oligonucleotides of Table 10.

In one embodiment, the oligonucleotide-antibody conjugate has a DARranging from about 1 to about 20, from about 1 to about 10, from about 1to about 8, from about 1 to about 4, or from about 1 to about 2. Inanother embodiment, the oligonucleotide-antibody conjugate has a DAR ofabout 1, about 2, about 3, about 4, about 5, about 6, about 7, or about8.

In some embodiments, the conjugate comprises one or more, two or more,three or more, four or more, five or more, six or more, seven or more,eight or more, nine or more, ten or more, or twenty or more Q-tagpeptides. In some embodiments, the conjugate comprises one, two, three,four, five, six, seven, eight, nine, ten, or twenty Q-tag peptides. Insome embodiments, the conjugate has 2 Q-tag peptides. In someembodiments, the conjugate comprises one or more, two or more, three ormore, four or more, five or more, six or more, seven or more, eight ormore, nine or more, ten or more, or twenty or more immunomodulatingoligonucleotides. In some embodiments, the conjugate comprises one, two,three, four, five, six, seven, eight, nine, ten, or twentyimmunomodulating oligonucleotides. In some embodiments, the conjugatehas one immunomodulating oligonucleotide. An exemplary conjugate isshown in FIGS. 16A-16D.

In one aspect, the oligonucleotide in the oligonucleotide-antibodyconjugate is an immunomodulating (e.g., immunostimulating)polynucleotide. In certain embodiments, the immunomodulatingpolynucleotide comprises a 5-modified uridine or 5-modified cytidine. Incertain embodiments, the inclusion of 5-modified uridine (e.g.,5-ethynyl-uridine) at the 5′-terminus of the immunomodulatingpolynucleotide (e.g., among the two 5′-terminal nucleosides) enhancesthe immunomodulating properties of the polynucleotide. In certainembodiments, the immunomodulating polynucleotide is shorter (e.g.,comprising a total of from about 6 to about 16 nucleotides or from about12 to about 14 nucleotides) than a typical CpG ODN, which is from 18 to28 nucleotides in length. In certain embodiments, the shorterimmunomodulating polynucleotide (e.g., those comprising a total of fromabout 6 to about 16 nucleotides or from about 12 to about 14nucleotides) retains the immunomodulating activity of a longer, typicalCpG ODN; or exhibits higher immunomodulating activity (e.g., as measuredby NFκB activation or by the changes in the expression levels of cellsurface markers of activation or function such as CD40, HLADR, CD69 orCD80 or by the changes in the levels of at least one cytokine (e.g.,IL-6 or IL-10), as compared to the longer CpG ODN. In certainembodiments, the immunomodulating polynucleotide comprises an abasicspacer. In certain embodiments, the immunomodulating polynucleotidecomprises an internucleoside phosphotriester.

In certain embodiments, the immunomodulating polynucleotide providedherein exhibits stability (e.g., stability against nucleases) that issuperior to that of a CpG ODN containing mostly internucleosidephosphate (e.g., more than 50% of internucleoside phosphates) withoutsubstantially sacrificing its immunostimulating activity. This effectcan be achieved, e.g., by incorporating at least 50% (e.g., at least70%) internucleoside phosphorothioates or phosphorodithioates or throughthe inclusion of internucleoside phosphotriesters and/or internucleosideabasic spacers. Phosphotriesters and abasic spacers are also convenientfor conjugation to a targeting moiety. Phosphate-based phosphotriestersand abasic spacers can also be used for reduction of off-targetactivity, relative to polynucleotides with fully phosphorothioatebackbones. Without wishing to be bound by theory, this effect may beachieved by reducing self-delivery without disrupting targetingmoiety-mediated delivery to target cells. Accordingly, a polynucleotideprovided herein can include about 15 or fewer, about 14 or fewer, about13 or fewer, about 12 or fewer, about 11 or fewer, or about 10 or fewercontiguous internucleoside phosphorothioates. For example, animmunostimulating polynucleotide comprising a total of from about 12 toabout 16 nucleosides can contain about 10 or fewer contiguousinternucleoside phosphorothioates.

The immunostimulating polynucleotide provided herein can contain a totalof about 50 or fewer, about 30 or fewer, about 28 or fewer, or about 16or fewer nucleosides. The immunostimulating polynucleotide can contain atotal of at least 6, about 10 or more, or about 12 or more nucleosides.For example, the immunostimulating polynucleotide can contain a total offrom about 6 to about 30, from about 6 to about 28, from about 6 toabout 20, from about 6 to about 16, from about 10 to about 20, fromabout 10 to about 16, from about 12 to about 28, from about 12 to about20, or from about 12 to about 16 nucleosides.

In certain embodiments, the immunostimulating polynucleotide comprisesone or more phosphotriesters (e.g., internucleoside phosphotriesters)and/or phosphorothioates (e.g., from about 1 to about 6 or from about 1to about 4), e.g., at one or both termini (e.g., within the six5′-terminal nucleosides or the six 3′-terminal nucleosides). Theinclusion of one or more internucleoside phosphotriesters and/orphosphorothioates can enhance the stability of the polynucleotide byreducing the rate of exonuclease-mediated degradation.

In certain embodiments, the immunostimulating polynucleotide comprises aphosphotriester or a terminal phosphodiester, where the phosphotriesteror the terminal phosphodiester comprises a linker bonded to a targetingmoiety or a conjugating group and optionally to one or more (e.g., fromabout 1 to about 6) auxiliary moieties. In certain embodiments, theimmunostimulating polynucleotide comprises only one linker. In certainembodiments, the immunostimulating polynucleotide comprises only oneconjugating group.

The polynucleotide provided herein can be a hybridized polynucleotideincluding a strand and its partial or whole complement. The hybridizedpolynucleotides can have at least 6 complementary base pairings (e.g.,about 6, about 7, about 8, about 9, about 10, about 11, about 12, about13, about 14, about 15, about 16, about 17, about 18, about 19, about20, about 21, about 22, or about 23), up to the total number of thenucleotides present in the included shorter strand. For example, thehybridized portion of the hybridized polynucleotide can contain about 6,about 7, about 8, about 9, about 10, about 11, about 12, about 13, about14, about 15, about 16, about 17, about 18, about 19, about 20, about21, about 22, or about 23 base pairs.

In one aspect, the oligonucleotide in the oligonucleotide-antibodyconjugate comprises one or more CpG sites. In some embodiments, theoligonucleotide comprises at least 1, at least 2, or at least 3 CpGsites. In some embodiments, the oligonucleotide is an antisenseoligonucleotide As used herein, a “modified nucleotide” is a nucleotideother than a ribonucleotide (2′-hydroxyl nucleotide). In someembodiments, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 97%, at least 98%, at least 99%, or 100% of thenucleotides are modified nucleotides. As used herein, modifiednucleotides include, but are not limited to, deoxyribonucleotides,nucleotide mimics, abasic nucleotides, 2′-modified nucleotides, 3′ to 3′linkages (inverted) nucleotides, non-natural base-comprisingnucleotides, bridged nucleotides, peptide nucleic acids (PNAs),2′,3′-seco nucleotide mimics (unlocked nucleobase analogues), lockednucleotides, 3′-O-methoxy (2′ internucleoside linked) nucleotides,2′-F-Arabino nucleotides, 5′-Me, 2′-fluoro nucleotide, morpholinonucleotides, vinyl phosphonate deoxyribonucleotides, vinyl phosphonatecontaining nucleotides, and cyclopropyl phosphonate containingnucleotides (cPrpN). The 2′-modified nucleotides (i.e. a nucleotide witha group other than a hydroxyl group at the 2′ position of thefive-membered sugar ring) include, but are not limited to, 2′-O-alkylnucleotides, 2′-deoxy-2′-halo nucleotides, 2′-deoxy nucleotides,2′-methoxyethyl (2′-O-2-methoxylethyl) nucleotides, 2′-aminonucleotides, 2′aminoalkyl nucleotides, and 2′-alkyl nucleotides. In someembodiments, modified nucleotide is selected from the group consistingof 5-bromo-2′-O-methyluridine, 5-bromo-2′-deoxyuridine,2′-O-methyluridine, 2′-deoxyuridine, 2′-O-methylthymidine,2′-O-methylcytidine, 2′-O-(2-methoxyethyl)thymidine and8-oxo-7,8-dihydro-2′-deoxyguanosine. It is not necessary for allpositions in a given compound to be uniformly modified. Conversely, morethan one modification may be incorporated in a single oligonucleotide oreven in a single nucleotide thereof. The oligonucleotides may besynthesized and/or modified by methods known in the art. Modification atone nucleotide is independent of modification at another nucleotide.

Modified nucleobases include synthetic and natural nucleobases, such as5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6substituted purines, (e.g., 2-aminopropyladenine, 5-propynyluracil, or5-propynylcytosine), 5-methylcytosine (5-Me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-alkyl (e.g.,6-methyl, 6-ethyl, 6-isopropyl, or 6-n-butyl) derivatives of adenine andguanine, 2-alkyl (e.g., 2-methyl, 2-ethyl, 2-isopropyl, or 2-n-butyl)and other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine, 2-thiocytosine, 5-halouracil (e.g., 5-bromouracil and5-iodouracil), cytosine, 5-propynyl uracil, 5-propynyl cytosine, 6-azouracil, 6-azo cytosine, 6-azo thymine, 5-uracil (pseudouracil),4-thiouracil, 8-halo, 8-amino, 8-sulfhydryl, 8-thioalkyl, 8-hydroxyl,8-oxo and other 8-substituted adenines and guanines, 5-halo (e.g.,5-bromo and 5-iodo), 5-trifluoromethyl, and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, and3-deazaadenine.

In some embodiments, one or more nucleotides of the oligonucleotide arelinked by non-standard linkages or backbones (e.g., modifiedinternucleoside linkages or modified backbones). In some embodiments, amodified internucleoside linkage is a non-phosphate-containing covalentinternucleoside linkage. Modified internucleoside linkages or backbonesinclude, but are not limited to, 5′-phosphorothioate groups, chiralphosphorothioates, thiophosphates, phosphorodithioates,phosphotriesters, aminoalkyl-phosphotriesters, alkyl phosphonates (e.g.,methyl phosphonates or 3′-alkylene phosphonates), chiral phosphonates,phosphinates, phosphoramidates (e.g., 3′-amino phosphoramidate,aminoalkylphosphoramidates, or thionophosphoramidates),thionoalkyl-phosphonates, thionoalkylphosphotriesters, morpholinolinkages, boranophosphates having normal 3′-5′ linkages, 2′-5′ linkedanalogs of boranophosphates, or boranophosphates having invertedpolarity wherein the adjacent pairs of nucleoside units are linked 3′-5′to 5′-3′ or 2′-5′ to 5′-2′. In some embodiments, a modifiedinternucleoside linkage or backbone lacks a phosphorus atom. Modifiedinternucleoside linkages lacking a phosphorus atom include, but are notlimited to, short chain alkyl or cycloalkyl inter-sugar linkages, mixedheteroatom and alkyl or cycloalkyl inter-sugar linkages, or one or moreshort chain heteroatomic or heterocyclic inter-sugar linkages. In someembodiments, modified internucleoside backbones include, but are notlimited to, siloxane backbones, sulfide backbones, sulfoxide backbones,sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, alkene-containing backbones,sulfamate backbones, methyleneimino and methylenehydrazino backbones,sulfonate and sulfonamide backbones, amide backbones, and otherbackbones having mixed N, O, S, and CH₂ components.

In some embodiments, the oligonucleotide comprises at least 2, at least3, at least 4, at least 5, at least 6, at least 7, at least 8, at least9, at least 10, at least 11, at least 12, at least 13, at least 14, orat least 15 phosphorothioate linkages. In some embodiments, theoligonucleotide comprises at least 2, at least 3, at least 4, at least5, at least 6, at least 7, at least 8, at least 9, at least 10, at least11, at least 12, at least 13, at least 14, or at least 15phosphorodithioate linkages. In some embodiments, the oligonucleotidecomprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15phosphorothioate linkages. In some embodiments, the oligonucleotidecomprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15phosphorodithioate linkages. In some embodiments, the phosphorothioateinternucleoside linkages or phosphorodithioate internucleoside linkagesare between the nucleotides at positions 1-3, 2-4, 3-5, 4-6, 4-5, 6-8,7-9, 8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19,18-20 or 19-21 from the 5′ end of the oligonucleotide. In someembodiments, the oligonucleotide comprises one or more modifiednucleotides and one or more modified internucleoside linkages.

In some embodiments, the oligonucleotide comprises a terminal cap. Insome embodiments, the terminal cap is at the 3′ end of theoligonucleotide. In some embodiments, the terminal cap is at the 5′ endof the oligonucleotide. In some embodiments, the terminal cap is at the5′ end and 3′ end of the oligonucleotide. The term “terminal cap” canalso be referred to as “cap,” and has meaning generally accepted in theart. For example, the term refers to a moiety, which can be a chemicallymodified nucleotide or non-nucleotide that can be incorporated at one ormore termini of one or more nucleic acid molecules of the invention.These terminal modifications can protect the nucleic acid molecule fromexonuclease degradation, and can help in delivery and/or localizationwithin a cell. In non-limiting examples, the cap includes, but is notlimited to a polymer; a ligand; locked nucleic acid (LNA); glyceryl; anabasic ribose residue; inverted deoxy abasic residue; an invertednucleotide; 4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl)nucleotide; 5′-mercapto moieties; 4′-thio nucleotide; carbocyclicnucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides;alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage;threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; acyclic3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide;3′-3′-inverted nucleotide moiety; 3′-3′-inverted abasic moiety;3′-2′-inverted nucleotide moiety; 3′-2′-inverted abasic moiety;1,4-butanediol phosphate; 3′-phosphoramidate; hexylphosphate; aminohexylphosphate; 3′-phosphate; 3′-phosphorothioate; 5′-5′-inverted nucleotidemoiety; 5′-5′-inverted abasic moiety; 5′-phosphoramidate;5′-phosphorothioate; 1,4-butanediol phosphate; 5′-amino; bridging and/ornonbridging 5′-phosphoramidate; phosphorothioate and/orphosphorodithioate; or bridging or non-bridging methylphosphonatemoiety. In some embodiments, the oligonucleotide comprises one or moreterminal cap molecules. In some embodiments, [N] is a 3′ terminal cap.In some embodiments, the 3′ terminal cap isO-(3-hydroxypropyl)phosphorothioate.

In some embodiments, the oligonucleotide is about 10-30, about 10-15,about 15-20, about 20-25, about 25-30, about 15-25 nucleotides inlength. In some embodiments, the oligonucleotide is about 18, 19, 20,21, 22, 23, 24 or 25 nucleotides in length.

In another aspect, the oligonucleotide of the conjugate is:

wherein

b and c are each independently an integer from 1 to 25; with the provisothat the sum of b and c is at least 5;

indicates the point of attachment of the immunomodulatingoligonucleotide P to the rest of the conjugate;

X^(5′) is a 5′ terminal nucleoside having the structure

X^(3′) is a 3′ terminal nucleoside having the structure

-   -   Y^(PTE) is an internucleoside phosphotriester having the        structure

wherein * indicates the points of attachment to the rest of theoligonucleotide and

indicates the point of attachment to the linker L, or, if L is absent,

indicates the point of attachment to the Q tag peptide Q at theglutamine residue via an amide bond;

Y^(3′) is a terminal phosphotriester having the structure

each X^(N) is independently a nucleoside having the structure

each Y^(N) is independently an internucleoside linker having thestructure

wherein each B^(N) is independently a modified or unmodified nucleobase;

each R^(N) is independently —H or —O—C₁₋₄-alkyl, wherein the C₁₋₄-alkylof the —O—C₁₋₄-alkyl is optionally further substituted by —O—C₁₋₄-alkyl;

B^(5′) and B^(3′) are independently a modified or unmodified nucleobase;

R^(5′) and R^(3′) are independently —H or —O—C₁-C₄-alkyl, wherein theC₁₋₄-alkyl of the —O—C₁₋₄-alkyl is optionally further substituted by—O—C₁₋₄-alkyl;

each T₁ is independently O or S;

each T₂ is independently O⁻ or S⁻; and

T₃ is a group comprising an oligoethylene glycol moiety; and

R¹ is C₁₋₄-alkylene-hydroxy.

In certain embodiments, the oligonucleotide comprises a nucleotide witha modified nucleobase. In some embodiments, B^(5′) is a modifiednucleobase. In other embodiments, B^(3′) is a modified nucleobase. Insome embodiments, B^(5′) is an unmodified nucleobase. In otherembodiments, B^(3′) is an unmodified nucleobase. In still otherembodiments, at least one B^(N) is a modified nucleobase.

In certain embodiments, b is an integer ranging from about 1 to about15. In certain embodiments, b is an integer of about 1, about 2, about3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about11, about 12, about 13, about 14, or about 15. In certain embodiments, bis an integer of about 3, about 4, about 11, or about 14. In certainembodiments, b is an integer of about 3. In certain embodiments, b is aninteger of about 4. In certain embodiments, b is an integer of about 11.In certain embodiments, b is an integer of about 14.

In certain embodiments, c is an integer ranging from about 0 to about10. In certain embodiments, c is an integer of about 0, about 1, about2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, orabout 10. In certain embodiments, c is an integer of about 0 or about 8.In certain embodiments, c is an integer of about 0. In certainembodiments, c is an integer of about 8.

In certain embodiments, b is an integer of about 3 and c is an integerof about 8. In certain embodiments, b is an integer of about 4 and c isan integer of about 8. In certain embodiments, b is an integer of about11 and c is an integer of about 0. In certain embodiments, b is aninteger of about 14 and c is an integer of about 0.

In certain embodiments, b and c together in total are ranging from about5 to about 20. In certain embodiments, b and c together in total areranging from about 5 to about 15. In certain embodiments, b and ctogether in total are about 5, about 6, about 7, about 8, about 9, about10, about 11, about 12, about 13, about 14, or about 15. In certainembodiments, b and c together in total are about 8, about 9, about 10,about 11, about 12, about 13, or about 14. In certain embodiments, b andc together in total are about 11. In certain embodiments, b and ctogether in total are about 12. In certain embodiments, b and c togetherin total are about 14.

In certain embodiments, each X^(N) is independently a2′-deoxyribonucleoside or a 2′-modified ribonucleoside. In certainembodiments, each X^(N) is independently 2′-deoxyadenosine (A),2′-deoxyguanosine (G), 2′-deoxycytidine (C), a 5-halo-2′-deoxycytidine,2′-deoxythymidine (T), 2′-deoxyuridine (U), a 5-halo-2′-deoxyuridine, a2′-fluororibonucleoside, a 2′-methoxyribonucleoside, or a2′-(2-methoxyethoxy)ribonucleoside. In certain embodiments, each X^(N)is independently a 2′-deoxyribonucleoside. In certain embodiments, eachX^(N) is independently 2′-deoxyadenosine, 2′-deoxyguanosine,2′-deoxycytidine, a 5-halo-2′-deoxycytidine, 2′-deoxythymidine,2′-deoxyuridine, or a 5-halo-2′-deoxyuridine. In certain embodiments,each X^(N) is independently 2′-deoxyadenosine, 2′-deoxyguanosine,2′-deoxycytidine, 2′-deoxythymidine, 5-bromo-2′-deoxyuridine, or5-iodo-2′-deoxyuridine.

In certain embodiments, X^(3′) is a 2′-deoxyribonucleoside or a2′-modified ribonucleoside. In certain embodiments, X^(3′) is a2′-deoxyribonucleoside. In certain embodiments, X^(3′) is2′-deoxyadenosine, 2′-deoxyguanosine, 2′-deoxycytidine, a5-halo-2′-deoxycytidine, 2′-deoxythymidine, 2′-deoxyuridine, a5-halo-2′-deoxyuridine, a 2′-fluororibonucleoside, a2′-methoxyribonucleoside, or a 2′-(2-methoxyethoxy)ribonucleoside. Incertain embodiments, X^(3′) is 2′-deoxyadenosine, 2′-deoxyguanosine,2′-deoxycytidine, a 5-halo-2′-deoxycytidine, 2′-deoxythymidine,2′-deoxyuridine, or a 5-halo-2′-deoxyuridine. In certain embodiments,X^(3′) is 2′-deoxythymidine. In certain embodiments, X^(3′) is a2′-deoxyribonucleoside with a substituted pyrimidine base. In certainembodiments, X^(3′) is a 2′-deoxyribonucleoside with a 5-substitutedpyrimidine base. In certain embodiments, X^(3′) is 2′-deoxythymidine, a5-halo-2′-deoxycytidine, or a 5-halo-2′-deoxyuridine. In certainembodiments, X^(3′) is 2′-deoxythymidine, 5-bromo-2′-deoxycytidine,5-iodo-2′-deoxycytidine, 5-bromo-2′-deoxyuridine, or5-iodo-2′-deoxyuridine. In certain embodiments, X^(3′) is2′-deoxythymidine, 5-bromo-2′-deoxyuridine, or 5-iodo-2′-deoxyuridine.In certain embodiments, X^(3′) is a terminal nucleotide comprising a 3′capping group. In certain embodiments, the 3′ capping group is aterminal phosphoester. In certain embodiments, the 3′ capping group is3-hydroxyl-propylphosphoryl (i.e., —P(O₂)—OCH₂CH₂CH₂OH).

In certain embodiments, X^(5′) is a 2′-deoxyribonucleoside or a2′-modified ribonucleoside. In certain embodiments, X^(5′) is a2′-deoxyribonucleoside. In certain embodiments, X^(5′) is2′-deoxyadenosine, 2′-deoxyguanosine, 2′-deoxycytidine, a5-halo-2′-deoxycytidine, 2′-deoxythymidine, 2′-deoxyuridine, a5-halo-2′-deoxyuridine, a 2′-fluororibonucleoside, a2′-methoxyribonucleoside, or a 2′-(2-methoxyethoxy)ribonucleoside. Incertain embodiments, X^(5′) is 2′-deoxyadenosine, 2′-deoxyguanosine,2′-deoxycytidine, a 5-halo-2′-deoxycytidine, 2′-deoxythymidine,2′-deoxyuridine, or a 5-halo-2′-deoxyuridine. In certain embodiments,X^(5′) is a 2′-deoxyribonucleoside with a substituted pyrimidine base.In certain embodiments, X^(5′) is a 2′-deoxyribonucleoside with a5-substituted pyrimidine base. In certain embodiments, X^(5′) is2′-deoxythymidine, a 5-halo-2′-deoxycytidine, or a5-halo-2′-deoxyuridine. In certain embodiments, X^(5′) is a5-halo-2′-deoxycytidine. In some embodiments, X^(5′) is a2′-deoxyuridine, a 5-halo-2′-deoxyuridine, 2′-methoxyuridine, or a5-halo-2′-methoxyuridine. In certain embodiments, X^(5′) is a5-halo-2′-deoxyuridine. In certain other embodiments, X^(5′) is a2′-deoxyuridine. In certain embodiments, X^(5′) is a5-halo-2′-methoxyuridine. In certain other embodiments, X^(5′) is a2′-methoxyuridine. In certain embodiments, X^(5′) is 2′-deoxythymidine,5-bromo-2′-deoxycytidine, 5-iodo-2′-deoxycytidine,5-bromo-2′-deoxyuridine, or 5-iodo-2′-deoxyuridine. In certainembodiments, X^(5′) is 2′-deoxythymidine, 5-bromo-2′-deoxyuridine, or5-iodo-2′-deoxyuridine. In certain embodiments, X^(5′) is5-bromo-2′-deoxyuridine. In certain embodiments, X^(5′) is5-iodo-2′-deoxyuridine. In certain embodiments, X^(5′) has a3′-phosphorothioate group. In certain embodiments, X^(5′) has a3′-phosphorothioate group with a chirality of Rp. In certainembodiments, X^(5′) has a 3′-phosphorothioate group with a chirality ofSp.

In certain embodiments, Y^(PTE) is an internucleosidephosphothiotriester.

In some embodiments, Y^(PTE) is

wherein Z is O or S; d is an integer ranging from about 0 to about 50;the two

on the right side of the structure indicate the points of attachment tothe oligonucleotide P; and the

on the left side of the structure indicates the point of attachment tothe rest of the conjugate. In certain embodiments, Z is O. In certainembodiments, Z is S. In certain embodiments, d is an integer rangingfrom about 0 to about 10. In certain embodiments, d is an integerranging from about 0 to about 5. In certain embodiments, d is an integerof about 0, about 1, about 2, about 3, about 4, or about 5. In certainembodiments, d is an integer of about 0, about 1, or about 3.

In some embodiments, Y^(PTE) is

wherein Z is O or S; d is an integer ranging from about 0 to about 50;the two

on the right side of the structure indicate the points of attachment tothe oligonucleotide P; and the

on the left side of the structure indicates the point of attachment tothe rest of the conjugate. In certain embodiments, Z is O. In certainembodiments, Z is S. In certain embodiments, d is an integer rangingfrom about 0 to about 10. In certain embodiments, d is an integerranging from about 0 to about 5. In certain embodiments, d is an integerof about 0, about 1, about 2, about 3, about 4, or about 5. In certainembodiments, d is an integer of about 0, about 1, or about 3.

In certain embodiments, the oligonucleotide comprises one additionalinternucleoside phosphotriester. In one embodiment, the additionalinternucleoside phosphotriester is a C₁₋₆ alkylphosphotriester. Inanother embodiment, the additional internucleoside phosphotriester isethylphosphotriester.

In certain embodiments, the oligonucleotide comprises one5-halo-2′-deoxyuridine. In one embodiment, the 5-halo-2′-deoxyuridine is5-fluoro-2′-deoxyuridine, 5-bromo-2′-deoxyuridine, or5-iodo-2′-deoxyuridine. In another embodiment, the5-halo-2′-deoxyuridine is 5-bromo-2′-deoxyuridine or5-iodo-2′-deoxyuridine. In yet another embodiment, the5-halo-2′-deoxyuridine is 5-fluoro-2′-deoxyuridine. In yet anotherembodiment, the 5-halo-2′-deoxyuridine is 5-bromo-2′-deoxyuridine. Instill another embodiment, the 5-halo-2′-deoxyuridine is5-iodo-2′-deoxyuridine.

In certain embodiments, the oligonucleotide comprises three or more2′-deoxycytidines. In certain embodiments, the oligonucleotide comprisesthree 2′-deoxycytidines.

In certain embodiments, the oligonucleotide comprises four or more2′-deoxyguanosines. In certain embodiments, the oligonucleotidecomprises four 2′-deoxyguanosines.

In certain embodiments, the oligonucleotide comprises three2′-deoxycytidines and four 2′-deoxyguanosines. In certain embodiments,the oligonucleotide comprises one, two, or three CG dinucleotides. Incertain embodiments, the oligonucleotide comprises three CGdinucleotides.

In certain embodiments, the oligonucleotide comprises three or more2′-deoxythymidines. In certain embodiments, the oligonucleotidecomprises three, four, five, six, seven, or eight 2′-deoxythymidines. Incertain embodiments, the oligonucleotide comprises three, four, five, oreight 2′-deoxythymidines.

In certain embodiments, the oligonucleotide does not comprise a2′-deoxyadenosine. In certain embodiments, the oligonucleotide comprisesone or two 2′-deoxyadenosines.

In certain embodiments, the oligonucleotide has a length ranging fromabout 5 to about 20 or from about 6 to about 15. In certain embodiments,the oligonucleotide has a length of about 6, about 7, about 8, about 9,about 10, about 11, about 12, about 13, about 14, or about 15. Incertain embodiments, the oligonucleotide has a length of about 10, about11, about 12, about 13, about 14, or about 15.

In certain embodiments, the oligonucleotide comprises one or moreinternucleoside phosphorothioates. In certain embodiments, all theinternucleoside phosphoesters in the oligonucleotide are internucleosidephosphorothioates. In certain embodiments, the oligonucleotide comprisesone or more chiral internucleoside phosphorothioates.

In certain embodiments, the oligonucleotides comprising a sequence ofN¹N²CGN³CG(T)_(x)GN⁴CGN⁵T (SEQ ID NO:174), or a stereoisomer, a mixtureof two or more diastereomers, a tautomer, or a mixture of two or moretautomers thereof, or a pharmaceutically acceptable salt, solvate, orhydrate thereof are as described in, for example, WO2018/189382 A1.

In one embodiment, the oligonucleotide comprises a sequence ofN¹N²CGN³CG(T)_(x)GN⁴CGN⁵T (SEQ ID NO:174), or a stereoisomer, a mixtureof two or more diastereomers, a tautomer, or a mixture of two or moretautomers thereof, or a pharmaceutically acceptable salt, solvate, orhydrate thereof; wherein:

x is an integer ranging from about 1 to about 4;

N¹ is absent or 2′-deoxythymidine;

N² is a 2′-deoxyribonucleotide with a modified nucleobase;

N³ is 2′-deoxyadenosine or 2′-deoxythymidine, each optionally comprisinga 3′-phosphotriester;

N⁴ is 2′-deoxyadenosine or 2′-deoxythymidine;

N⁵ is 2′-deoxythymidine optionally comprising a 3′-phosphotriester; and

C is 2′-deoxycytidine and G is 2′-deoxyguanosine.

In certain embodiments, in N¹N²CGN³CG(T)_(x)GN⁴CGN⁵T (SEQ ID NO:174), xis an integer of about 1, about 2, about 3, or about 4. In certainembodiments, in N¹N²CGN³CG(T)_(x)GN⁴CGN⁵T (SEQ ID NO:174), x is aninteger of about 1. In certain embodiments, in N¹N²CGN³CG(T)_(x)GN⁴CGN⁵T(SEQ ID NO:174), x is an integer of about 4.

In certain embodiments, in N¹N²CGN³CG(T)_(x)GN⁴CGN⁵T (SEQ ID NO:174), N¹is absent. In certain embodiments, in N¹N²CGN³CG(T)_(x)GN⁴CGN⁵T (SEQ IDNO:174), N¹ is 2′-deoxythymidine.

In certain embodiments, in N¹N²CGN³CG(T)_(x)GN⁴CGN⁵T (SEQ ID NO:174), N²is a 2′-deoxyribonucleotide with a substituted pyrimidine base. Incertain embodiments, in N¹N²CGN³CG(T)_(x)GN⁴CGN⁵T (SEQ ID NO:174), N² isa 2′-deoxyribonucleotide with a 5-substituted pyrimidine base. Incertain embodiments, in N¹N²CGN³CG(T)_(x)GN⁴CGN⁵T (SEQ ID NO:174), N² isa 5-halo-2′-deoxycytidine or a 5-halo-2′-deoxyuridine. In certainembodiments, in N¹N²CGN³CG(T)_(x)GN⁴CGN⁵T (SEQ ID NO:174), N² is5-bromo-2′-deoxyuridine or 5-iodo-2′-deoxyuridine.

In certain embodiments, in N¹N²CGN³CG(T)_(x)GN⁴CGN⁵T (SEQ ID NO:174), N³is 2′-deoxyadenosine comprising a 3′-phosphotriester. In certainembodiments, in N¹N²CGN³CG(T)_(x)GN⁴CGN⁵T (SEQ ID NO:174), N³ is2′-deoxythymidine. In certain embodiments, in N¹N²CGN³CG(T)_(x)GN⁴CGN⁵T(SEQ ID NO:174), N³ is 2′-deoxythymidine comprising a3′-phosphotriester.

In certain embodiments, in N¹N²CGN³CG(T)_(x)GN⁴CGN⁵T (SEQ ID NO:174), N⁴is 2′-deoxyadenosine. In certain embodiments, inN¹N²CGN³CG(T)_(x)GN⁴CGN⁵T (SEQ ID NO:174), N⁴ is 2′-deoxythymidine.

In certain embodiments, in N¹N²CGN³CG(T)_(x)GN⁴CGN⁵T (SEQ ID NO:174), N⁵is 2′-deoxythymidine. In certain embodiments, inN¹N²CGN³CG(T)_(x)GN⁴CGN⁵T (SEQ ID NO:174), N⁵ is 2′-deoxythymidinecomprising a 3′-phosphotriester.

In certain embodiments, the oligonucleotide of N¹N²CGN³CG(T)_(x)GN⁴CGN⁵T(SEQ ID NO:174) comprises one or more internucleoside phosphorothioatesor phosphorotdithioates. In certain embodiments, the oligonucleotide ofN¹N²CGN³CG(T)_(x)GN⁴CGN⁵T (SEQ ID NO:174) comprises at least one chiralinternucleoside phosphorothioate or phosphorotdithioates. In certainembodiments, the oligonucleotide of N¹N²CGN³CG(T)_(x)GN⁴CGN⁵T (SEQ IDNO:174) comprises at least one chiral phosphorotdithioates. In certainembodiments, the oligonucleotide of N¹N²CGN³CG(T)_(x)GN⁴CGN⁵T (SEQ IDNO:174) is an oligonucleotide sequence as described in, for example,WO2018/189382 A1.

In certain embodiments, the oligonucleotide provided herein is animmunostimulating polynucleotide. In certain embodiments, theoligonucleotide provided herein functions as a PAMS. In certainembodiments, the oligonucleotide provided herein activates innate immuneresponse or stimulates the adaptive immune response by triggering TLR9signaling. In certain embodiments, the oligonucleotide provided hereinis a TLR9 agonist.

In certain embodiments, the oligonucleotide provided herein is CpGoligonucleotide, comprising a modification including 5-halouridine or5-alkynyluridine, or a truncated version thereof (e.g., those comprisinga total of about 6 to about 16 nucleosides). In certain embodiments, thetruncated oligonucleotide provided herein comprises a truncatedoligonucleotide sequence, from which one or more 3′-terminal nucleotidesare eliminated or one or more of the intra-sequence nucleotidesexcised).

In certain embodiments, the oligonucleotide provided herein comprises atleast one immunostimulating sequence (ISS). In certain embodiments, theoligonucleotide provided herein comprises about 1, about 2, about 3, orabout 4 ISS. The ISS in immunostimulating polynucleotides is dependenton the targeted organism. The common feature of the ISS used in theoligonucleotide provided herein is the cytidine-p-guanosine sequence, inwhich p is an internucleoside phosphodiester (e.g., phosphate orphosphorothioate) or an internucleoside phosphotriester. In certainembodiments, cytidine and guanosine in the ISS each independentlycomprises 2′-deoxyribose. In certain embodiments, the oligonucleotideprovided herein comprises about 1, about 2, or about 3 human ISSs. Incertain embodiments, the human ISS is CG or NCG, where N is uridine,cytidine, or thymidine, or a modified uridine or cytidine; and G isguanosine or a modified guanosine. In certain embodiments, the modifieduridine or cytidine is a 5-halouridine (e.g., 5-iodouridine or5-bromouridine), a 5-alkynyluridine (e.g., 5-ethynyluridine or5-propynyluridine), 5-heteroaryluridine, or 5-halocytidine. In certainembodiments, the modified guanosine is 7-deazaguanosine. In certainembodiments, the human ISS is NCG, in one embodiment, N is5-halouridine. In certain embodiments, the human ISS is UCG, in oneembodiment, U is 5-alkynyluridine, and in another embodiment, U is5-ethynyluridine. In certain embodiments, the oligonucleotide providedherein targeting humans comprises an ISS within four contiguousnucleotides that include a 5′-terminal nucleotide. In certainembodiments, the oligonucleotide provided herein targeting humanscomprises a 5′-terminal ISS. In certain embodiments, the oligonucleotideprovided herein comprises a murine ISS. In certain embodiments, themurine ISS is a hexameric nucleotide sequence: Pu-Pu-CG-Py-Py, whereeach Pu is independently a purine nucleotide, and each Py isindependently a pyrimidine nucleotide.

In certain embodiments, the 5′-flanking nucleotides relative to CpG inthe oligonucleotide provided herein does not contain 2′-alkoxyriboses.In certain embodiments, the 5′-flanking nucleotides relative to CpG inthe oligonucleotide provided herein comprises only 2′-deoxyriboses assugars.

In certain embodiments, the oligonucleotide provided herein has (1) ahigh content of phosphorothioates or phosphorodithioates (e.g., at least50%, at least 60%, at least 70%, or at least 80% of nucleosides may belinked by phosphorothioates or phosphorodithioates); (2) absence ofpoly-G tails; (3) nucleosides in the oligonucleotide comprises2′-deoxyriboses or 2′-modified riboses (e.g., 2′-halo (e.g., 2′-fluoro,2′-bromo, or 2′-iodo) or optionally substituted 2′-alkoxy (e.g.,2′-methoxy)); and/or (4) the inclusion of 5′-terminal ISS that is NCG,in which N is uridine, cytidine, or thymidine, or a modified uridine orcytidine, and G is guanosine or a modified guanosine.

In certain embodiments, the oligonucleotide provided herein suppressesthe adaptive immune response by reducing activation of TLR9 signaling(e.g., through TLR9 antagonism). In certain embodiments, theimmunosuppressive polynucleotide provided herein comprises at least two2′-alkoxynucleotides that are 5′-flanking relative to CpG as describedby the formula of N¹-N²-CG, where N¹ and N² are each independently anucleotide containing 2′-alkoxyribose (e.g., 2′-methoxyribose).

In some embodiments, the oligonucleotide has the structure

wherein

and

indicate the points of attachment within the oligonucleotide;

each T¹ is independently O or S;

each T² is O⁻ or S⁻;

T³ is a group

wherein

indicates the point of attachment to L and wherein

indicates the point of attachment to the rest of the oligonucleotide;

Z is O or S;

U^(5′) is —H or halogen;

R^(5′) is —H or methoxy;

R^(c1) is —H or methoxy;

R^(g1), R^(g2), R^(g3), and R^(g4) are H or oxo, wherein

R^(3′) is methoxy;

R¹ is C₁₋₄-alkylene-hydroxy;

R² is —H or methyl; and

n is an integer from 0 to 2.

In other embodiments, the oligonucleotide has the structure

wherein

and

indicate the points of attachment within the oligonucleotide;

each T¹ is independently O or S;

each T² is O⁻ or S⁻;

T³ is a group

wherein

indicates the point of attachment to L and wherein

indicates the point of attachment to the rest of the oligonucleotide;

Z is O or S;

R^(5′) is —H or methoxy;

R^(c1) is —H or methoxy;

R^(g1), R^(g2), R^(g3), and R^(g4) are H or oxo, wherein

R^(3′) is methoxy;

R¹ is C₁₋₄-alkylene-hydroxy;

R² is —H or methyl; and

n is an integer from 0 to 2.

In still other embodiments, the oligonucleotide has the structure

wherein

and

indicate the points of attachment within the oligonucleotide;

T¹ is independently O or S;

each T² is O⁻ or S⁻;

T³ is a group

wherein

indicates the point of attachment to L and wherein

indicates the point of attachment to the rest of the oligonucleotide;

Z is O or S;

R^(5′) is —H or methoxy;

R^(c1) is —H or methoxy;

R^(g1), R^(g2), R^(g3), and R^(g4) are H or oxo, wherein

R^(3′) is methoxy;

R¹ is C₁₋₄-alkylene-hydroxy;

R² is —H or methyl; and

n is an integer from 0 to 2.

In some embodiments, the oligonucleotide comprises one or more ofunmodified sequences differing by 0, 1, 2 or 3 nucleobases from thesequences shown in Table 1. In some embodiments, the oligonucleotidecomprises one or more of modified sequences differing by 0, 1, 2 or 3nucleobases from the sequences shown in Table 2.

TABLE 1 Unmodified Oligonucleotides Unmodified SEQ Oligonucleotide IDSequence NO. (5′ >3′) 1 tucgtcgtgacgtt 2 ucgtcgtgtcgtt 129tcgtcgttttgtcgttttgtcgtt

TABLE 2 Modified Oligonucleotides SEQ Modified Oligonucleotide SequenceCmpd ID NO. (5′ → 3′) # 3

scsgstscsgstsgstscsgstsT-c3  1.1b 4

scs

stscsgstsgstscsgsts

-c3  2.1b 5

scsgstscs

stsgstscsgsts

-c3  2.2b 6

scsgstscsgsts

stscsgsts

-c3  2.3b 7

scsgstscsgstsgstscs

sts

-c3  2.4b 8

scsgstscsgstsgstscsgsts

-c3  3.1b 9

scsgstscsgstsgstscsgsts

s

-c3  3.2b 10

scsgstscsgstsgstscsgsts

s

s

-c3  3.3b 11

scsgstscsgstsgstscsgsts

-c3  4.1b 12

scsgstscsgstsgstscsgsts

s

-c3  4.2b 13

s

sgstscsgstsgstscsgsts

s

-c3  4.3b 14

scs2gs

scsgstsgstscsgsts

-c3  5.1a 15

scsgs2

scsgstsgstscsgsts

-c3  5.2a 16

scsgs

scs2gstsgstscsgsts

-c3  5.3a 17

scsgs

scsgs2tsgstscsgsts

-c3  5.4a 18

scsgs

scsgsts2gstscsgsts

-c3  5.5a 19

scsgs

scsgstsgs2tscsgsts

-c3  5.6a 20

scsgs

scsgstsgsts2csgsts

-c3  5.7a 21

scsgs

scsgstsgstscs2gsts

-c3  5.8a 22

scsgs

scsgstsgstscsgs2ts

-c3  5.9a 23

scsgs

scsgstsgstscsgsts2

-c3  5.10a 24

scsgs

scsgstsgstscsgsts 

s2-c3  5.11a 25

scs2gs

scsgstsgstscsgsts 

s2-c3  5.12a 26

s

sgs2tscsgstsgstscsgsts

s

-c3  6.1b 27

s

sgstscsgstsgsts2csgsts

s

-c3  6.2b 28

s

sgs2tscsgstsgsts2csgsts

s

-c3  6.3b 29

scs2gstscsgstsgstscsgsts

s

-c3  7.1b 30

scsgstscsgstsgs2tscsgsts

s

-c3  7.2b 31

scs2gstscsgstsgs2tscsgsts

s

-c3  7.3b 32

scsgstscs2gstsgstscsgsts

s

-c3  7.4b 33

scsgstscsgs2tsgstscsgsts

s

-c3  7.5b 34

scsgstscsgsts2gstscsgsts

s

-c3  7.6b 35

scsgstscsgstsgsts2csgsts

s

-c3  7.7b 36

scs2gstscsgstsgsts2csgsts

s

-c3  7.8b 37

scsgs2tscsgstsgstscsgsts

s

-c3  7.9b 38

scsgstscsgstsgstscs2gsts

s

-c3  7.10b 130 uscsgstscsgstsgstscsgstsT-c3  8.1b 131 uscs

stscsgstsgstscsgsts

-c3  9.1b 132 uscsgstscs

stsgstscsgsts

-c3  9.2b 133 uscsgstscsgsts

stscsgsts

-c3  9.3b 134 uscsgstscsgstsgstscsgsts

-c3  9.4b 135 uscsgstscsgstsgstscsgsts

-c3  10.1b 136 uscsgstscsgstsgstscsgsts

s

-c3  10.2b 137 uscsgstscsgstsgstscsgsts

s

s

-c3  10.3b 138

scsgstscsgstsgstscsgsts

-c3  12.1b 139

scsgstscsgstsgstscsgsts

s

-c3  12.2b 140

s

sgstscsgstsgstscsgsts

s

-c3  12.3b 141 uscs2gs

scsgstsgstscsgsts

-c3  13.1a 142 uscsgs2

scsgstsgstscsgsts

-c3  13.2a 143 uscsgs

scs2gstsgstscsgsts

-c3  13.3a 144 uscsgs

scsgs2tsgstscsgsts

-c3  13.4a 145 uscsgs

scsgsts2gstscsgsts

-c3  13.5a 146 uscsgs

scsgstsgs2tscsgsts

-c3  13.6a 147 uscsgs

scsgstsgsts2csgsts

-c3  13.7a 148 uscsgs

scsgstsgstscs2gsts

-c3  13.8a 149 uscsgs

scsgstsgstscsgs2ts

-c3  13.9a 150 uscsgs

scsgstsgstscsgsts2

-c3 13.10a 151 uscsgs

scsgstsgstscsgsts 

s2c3 13.11a 152 uscs2gs

scsgstsgstscsgsts 

s2-c3 13.12a 153

s

sgs2tscsgstsgstscsgsts

s

-c3  14.1b 154

s

sgstscsgstsgsts2csgsts

s

-c3  14.2b 155

s

sgs2tscsgstsgsts2csgsts

s

-c3  14.3b 156 uscs2gstscsgstsgstscsgsts

s

-c3  15.1b 157 uscsgstscsgstsgs2tscsgsts

s

-c3  15.2b 158 uscs2gstscsgstsgs2tscsgsts

s

-c3  15.3b 159 uscsgstscs2gstsgstscsgsts

s

-c3  15.4b 160 uscsgstscsgs2tsgstscsgsts

s

-c3  15.5b 161 uscsgstscsgsts2gstscsgsts

s

-c3  15.6b 162 uscsgs

scsgstsgsts2csgsts

s

-c3  15.7a 163 uscsgstscsgstsgsts2csgsts

s

-c3  15.7b 164 uscs2gstscsgstsgsts2csgsts

s

-c3  15.8b 165 uscsgs2tscsgstsgstscsgsts

s

-c3  15.9b 166 uscsgstscsgstsgstscs2gsts

s

-c3 15.10b *

: 5-Bromo-2′-deoxyuridine

: 8-oxo-7,8-dihydro-2′-deoxyguanosine

: 5-Bromo 2′-OMe uridine

: 2′-OMe-Cytidine

 : 2′-OMe-Thymidine

: 2′-OMe-Uridine u: 2′-deoxyuridine T: 2′-OMOE thymidine ts:phosphotriester linker-PEG₂₄-NH₂

following thymidine;

s:

phosphotriester linker following thymidine; Lower case: 2′-deoxynucleotide s: phosphorothioate linkage s2: phosphorodithioate linkagec3:

s2-c3:

In some embodiments, the oligonucleotide is functionalized with achemical tag for attachment to the linking moiety. In some embodiments,the chemical tag is attached to an inter-nucleoside linkage of theoligonucleotide. In some embodiments, the chemical tag is attached to a5′ inter-nucleoside linkage. In some embodiments, the chemical tag isattached to a 3′ inter-nucleoside linkage. In some embodiments, theinter-nucleoside linkage is a phosphorothioate linkage. In someembodiments, the inter-nucleoside linkage is a phosphorodithioatelinkage. In some embodiments, the chemical tag is closer to the 5′ endthan the 3′ end of the oligonucleotide. In some embodiments, thechemical tag is attached to a nucleobase.

Linking Moieties

In another aspect, the oligonucleotide is conjugated to the polypeptidevia a linking moiety. The length, rigidity and chemical composition ofthe linking moiety impact the conjugation reaction rates and thestability of the resulting conjugates. In some embodiments, the linkingmoiety comprises polyethylene glycol (PEG). In some embodiments, the PEGcontains about 10-50 ethylene glycol units. In some embodiments, thelinking moiety is an aliphatic chain.

For Formula (A), the linking moiety is represented by L. In someembodiments, the linker L comprises an oligoethylene glycol orpolyethylene glycol moiety. In certain embodiments, the linker L is agroup having the structure

wherein

indicates the point of attachment to Y^(PTE), and

indicates the point of attachment to the rest of the conjugate.

In other embodiments, the linker L is a group having the structure

wherein

indicates the point of attachment to Y^(PTE), and

indicates the point of attachment to the rest of the conjugate. In someembodiments, L¹ is absent. In some embodiments, L¹ is unsubstitutedalkyl. In some embodiments, L¹ is independently an unsubstituted C₁₋₆alkyl. In some embodiments, each L¹ is methyl or ethyl. In someembodiments, L¹ is independently a substituted alkyl. In someembodiments, L¹ is independently a substituted C₁₋₆ alkyl. In someembodiments, L¹ is C₁₋₆ alkyl substituted with one or more substituentsselected from the group consisting of alkoxy, acyl, acyloxy,alkoxycarbonyl, carbonylalkoxy, acylamino, amino, aminoacyl,aminocarbonylamino, aminocarbonyloxy, cycloalkyl, cycloalkenyl, cyano,azido, halo, hydroxyl, nitro, carboxyl, thiol, thioalkyl, alkyl,alkenyl, alkynyl, heterocyclyl, aminosulfonyl, sulfonylamino, sulfonyland oxo.

In some embodiments, L² is absent. In some embodiments, L² isunsubstituted or substituted alkyl.

In some embodiments, L³ is absent. In some embodiments, L³ is a linkermoiety. In some embodiments, the linker moiety is an unsubstituted orsubstituted alkyl. In some embodiments, the linker moiety isindependently an unsubstituted C₁₋₆ alkyl. In some embodiments, thelinker moiety is methyl or ethyl. In some embodiments, the linker moietyis independently a substituted alkyl. In some embodiments, the linkermoiety is independently a substituted C₁₋₆ alkyl. In some embodiments,the linker moiety is C₁₋₆ alkyl substituted with one or moresubstituents selected from the group consisting of alkoxy, acyl,acyloxy, alkoxycarbonyl, carbonylalkoxy, acylamino, amino, aminoacyl,aminocarbonylamino, aminocarbonyloxy, cycloalkyl, cycloalkenyl, cyano,azido, halo, hydroxyl, nitro, carboxyl, thiol, thioalkyl, alkyl,alkenyl, alkynyl, heterocyclyl, aminosulfonyl, sulfonylamino, sulfonyland oxo. In some embodiments, the linker moiety is an amino acidresidue. In some embodiments, the amino acid is selected from the groupconsisting of glycine, alanine, glutamic acid and proline. In someembodiments, the linker is methyl. In some embodiments, the linkermoiety is —R⁵C(O)R⁶NHR⁷—, wherein R⁵, and R⁷ are independently absent orunsubstituted or substituted alkyl and R⁶ is an amino acid residue. Insome embodiments, the amino acid is selected from the group consistingof glycine, alanine, glutamic acid and proline. In some embodiments, thelinker moiety is —R³C(O)NHR⁴—, wherein R³ and R⁴ are independentlyabsent or unsubstituted or substituted alkyl. In some embodiments, R³ ismethylene and R⁴ is —(CH₂)₄—. In some embodiments, R³ is methylene andR⁴ is absent. When more than one oligonucleotide (i.e., p=2), the two L¹can be different or same, the two L² can be different or same and thetwo L³ can be different or same.

In some embodiments, m is about 3-10, about 10-15, about 15-20, about20-25, about 25-30, about 5-16, about 15-30, about 15-25 or about 20-30.In some embodiments, m is 20, 21, 22, 23, 24 or 25.

Protein

In some embodiments, an oligonucleotide of the present disclosure isconjugated to a polypeptide or protein, e.g., an antibody. In someembodiments, the oligonucleotide is conjugated to an antibody via one ormore Q tags. In some embodiments, the Q tag comprises a glutamineresidue which is linked to the rest of the conjugate. In still furtherembodiments of the present aspect, which may be combined with any of thepreceding embodiments, each Q tag independently comprises or is apeptide sequence selected from the group consisting of SEQ ID NOs:39-55. In some embodiments, each Q tag independently comprises or is apeptide sequence selected from the group consisting of the peptidesequences of Table 3. In other embodiments of the present aspect, each Qtag independently comprises or is a peptide sequence selected from thegroup consisting of SEQ ID NOs: 40-55. In yet other embodiments, each Qtag independently comprises or is a peptide sequence selected from thegroup consisting of SEQ ID NOs: 47-49. In some embodiments, the Q-tagcomprises LLQGG (SEQ ID NO:172), GGGLLQGG (SEQ ID NO:173), RPQGF (SEQ IDNO:47), or RPQGFGPP (SEQ ID NO:49). In some embodiments, the Q-tagcomprises a peptide sequence RPQGF (SEQ ID NO:47). In certainembodiments, the Q-tag comprising a peptide sequence RPQGF (SEQ IDNO:47) is selected from the group consisting of RPQGF (SEQ ID NO:47),RPQGFPP (SEQ ID NO:48), and RPQGFGPP (SEQ ID NO:49).

In some embodiments, the protein is a protein fragment, a peptide or aFc-fusion protein. In some embodiments, the protein is an antibodyselected from a group consisting of a polyclonal antibody, a monoclonalantibody, a humanized antibody, a human antibody, a chimeric antibody,and an antibody fragment. In some embodiments, the antibody fragment isselected from the group consisting of Fab, Fab′, Fab′-SH, F(ab′)2, Fvfragments, scFv, single domain antibody, single heavy chain antibody andsingle light chain antibody. In some embodiments, the antibody is ahuman anti-IgG antibody. In some embodiments, the antibody is ananti-IgG1, anti-IgG2 or anti-IgG4 antibody. In some embodiments, theantibody is an anti-CD22 antibody (e.g., RFB4, EPRA, 10F4, m971). Insome embodiments, the antibody comprises a light chain variable domain(VL) and a heavy chain variable domain (VH). In some embodiments, VHcomprises the sequence SEQ ID NO: 56 and VL comprises the sequence SEQID NO: 57. In some embodiments, VH comprises the sequence SEQ ID NO: 58and VL comprises the sequence SEQ ID NO: 59. In some embodiments, VHcomprises the sequence SEQ ID NO: 60 and VL comprises the sequence SEQID NO: 61. In some embodiments, VH comprises the sequence SEQ ID NO: 62and VL comprises the sequence SEQ ID NO: 63.

In some embodiments, the antibody comprises a heavy chain variable (VH)domain and a light chain variable (VL) domain, wherein the VH domaincomprises CDR-H1, CDR-H2, and CDR-H3 sequences from a VH domain sequenceselected from the group consisting of:

(SEQ ID NO: 64) EVQLVESGGGLVQPGGSLRLSCAASGFAFSIYDMSWVRQAPGKGLEWVAYISSGGGTTYYPDTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHSGYGTHWGVLFAYWGRGTLVTVSS, (SEQ ID NO: 65)QVQLLESGGGVVQPGGSLRLSCAASGFAFSIYDMNWVRQAPGKGLEWVSAISSGGGTTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHSGYGTHWGVLFAYWGRGTLVTVSS, (SEQ ID NO: 66)EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYEMNWVRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHSGYGTHWGVLFAYWGRGTLVTVSS, and (SEQ ID NO: 67)QVQLQESGPGLVKPSDTLSLTCTVSGFAFSIYDMSWIRQPPGKGLEWIAYISSGGGTTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARHSGYGTHWGVLFAYWGRGTLVTVSS.

In some embodiments, the antibody comprises a heavy chain variable (VH)domain and a light chain variable (VL) domain, and wherein the VH domaincomprises an amino acid sequence selected from the group consisting of:

(SEQ ID NO: 64) EVQLVESGGGLVQPGGSLRLSCAASGFAFSIYDMSWVRQAPGKGLEWVAYISSGGGTTYYPDTVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCARHSGYGTHWGVLFAYWGRGTLVTVSS, (SEQ ID NO: 65) QVQLLESGGGVVQPGGSLRLSCAASGFAFSIYDMNWVRQAPGKGLEWVSAISSGGGTTYYADSVKGRFTI SRDNAKNSLYLQMNSLRAEDTAVYYCARHSGYGTHWGVLFAYWGRGTLVTVSS, (SEQ ID NO: 66) EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYEMNWVRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTI SRDNAKNSLYLQMNSLRAEDTAVYYCARHSGYGTHWGVLFAYWGRGTLVTVSS, and (SEQ ID NO: 67)QVQLQESGPGLVKPSDTLSLTCTVSGFAFSIYDMS WIRQPPGKGLEWIAYISSGGGTTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARIISGYGT IIWGVLFAYWGRGTLVTVSS.

In other embodiments, which may be combined with any of the foregoingembodiments, the antibody comprises a heavy chain variable (VH) domainand a light chain variable (VL) domain, wherein the VL domain comprisesCDR-L1, CDR-L2, and CDR-L3 sequences from a VL domain sequence selectedfrom the group consisting of:

(SEQ ID NO: 68) DIQMTQSPSSLSASVGDRVTITCRASQDIHGYLNWYQQKPGKAPKLLIYYTSILHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQGNTLPWTFGQ GTKLEIK,(SEQ ID NO: 69) DIQMTQSPSSVSASVGDRVTITCRASQDIHGYLAWYQQKPGKAPKLLIYYTSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNTLPWTFGQ GTKLEIK,(SEQ ID NO: 70) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNTLPWTFGQ GTKLEIK,(SEQ ID NO: 71) EIVLTQSPATLSLSPGERATLSCRASQDIHGYLNWYQQKPGQAPRLLIYYTSILHSGIPARFSGSGPGTDFTLTISSLEPEDFAVYYCQQGNTLPWTFGG GTKLEIK, and(SEQ ID NO: 72) DIVMTQTPLSLSVTPGQPASISCRASQDIFIGYLNWYQQKPGQSPQLLIYYTSILHSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCQQGNTLPWTFG GGTKLEIK.

In some embodiments, which may be combined with any of the foregoingembodiments, the antibody comprises an Fc region. In certainembodiments, the Fc region is a human Fc region selected from the groupconsisting of an IgG1 Fc region, an IgG2 Fc region, and an IgG4 Fcregion. In some embodiments, the Fc region is a wild-type human IgG1,IgG2, or IgG4 Fc region. In some embodiments, the Fc region is a humanFc region comprising one or more amino acid substitutions that reduce oreliminate one or more effector functions, as compared with the effectorfunction(s) of a human Fc region that lacks the amino acidsubstitution(s). In still yet further embodiments, the Fc region is: (a)a human IgG1 Fc region comprising L234A, L235A, and/or G237Asubstitutions, amino acid position numbering according to EU index; (b)a human IgG2 Fc region comprising A330S and/or P331S substitutions,amino acid position numbering according to EU index; or (c) a human IgG4Fc region comprising S228P and/or L235E substitutions, amino acidposition numbering according to EU index. In some embodiments, the Fcregion is a human Fc region comprising one or more amino acidsubstitutions that reduce or eliminate binding to human C1q, as comparedwith the binding of a human Fc region that lacks the amino acidsubstitution(s). In some embodiments, the Fc region is a human Fc regioncomprising one or more amino acid substitutions that reduce or eliminateantibody-dependent cellular cytotoxicity (ADCC), as compared with theADCC of a human Fc region that lacks the amino acid substitution(s).

Antibodies that target cell surface antigens can triggerimmunostimulatory and effector functions that are associated with Fcreceptor (FcR) engagement on immune cells. There are a number of Fcreceptors that are specific for particular classes of antibodies,including IgG (gamma receptors), IgE (eta receptors), IgA (alphareceptors) and IgM (mu receptors). Binding of the Fc region to Fcreceptors on cell surfaces can trigger a number of biological responsesincluding phagocytosis of antibody-coated particles (antibody-dependentcell-mediated phagocytosis, or ADCP), clearance of immune complexes,lysis of antibody-coated cells by killer cells (antibody-dependentcell-mediated cytotoxicity, or ADCC) and, release of inflammatorymediators, placental transfer, and control of immunoglobulin production.Additionally, binding of the C1 component of complement to antibodiescan activate the complement system. Activation of complement can beimportant for the lysis of cellular pathogens. However, the activationof complement can also stimulate the inflammatory response and can alsobe involved in autoimmune hypersensitivity or other immunologicaldisorders. Variant Fc regions with reduced or ablated ability to bindcertain Fc receptors are useful for developing therapeutic antibodiesand Fc-fusion polypeptide constructs which act by targeting, activating,or neutralizing ligand functions while not damaging or destroying localcells or tissues.

In some embodiments, an Fc domain can refer to a dimer of two Fc domainmonomers. In a wild-type Fc domain, two Fc domain monomers dimerize bythe interaction between the two CH₃ antibody constant domains, as wellas one or more disulfide bonds that form between the hinge domains ofthe two dimerized Fc domain monomers. In some embodiments, an Fc domainis mutated to lack effector functions, for example a “dead Fc domain.”In some embodiments, each of the Fc domain monomers in an Fc domainincludes amino acid substitutions in the CH2 antibody constant domain toreduce the interaction or binding between the Fc domain and an Fcreceptor, such as an Fcγ receptor (FcγR), an Fcα receptor (FcαR), or anFcε (FcεR).

The Fc domain is not involved directly in binding an antibody to itstarget, but can be involved in various effector functions, such asparticipation of the antibody in antibody-dependent cellular toxicity.In some embodiments, the Fc domain in an antibody or conjugate of thedisclosure comprises one or more amino acid substitutions, additions orinsertions, deletions, or any combinations thereof that lead todecreased effector function such as decreased antibody-dependentcell-mediated cytotoxicity (ADCC), decreased complement-dependentcytolysis (CDC), decreased antibody-dependent cell-mediated phagocytosis(ADCP), or any combinations thereof. In some embodiments, the antibodiesor conjugates of the disclosure are characterized by decreased binding(e.g., minimal binding or absence of binding) to a human Fc receptor anddecreased binding (e.g., minimal binding or absence of binding) tocomplement protein C1q. In some embodiments, the antibodies orconjugates of the disclosure are characterized by decreased binding(e.g., minimal binding or absence of binding) to human FcγRI, FcγRIIA,FcγRIIB, FcγRIIIB, FcγRIIIB, or any combinations thereof, and C1q. Toalter or reduce an antibody-dependent effector function, such as ADCC,CDC, ADCP, or any combinations thereof, in some embodiments, the Fcdomains in antibodies or conjugates of the disclosure are of the IgGclass and comprise one or more amino acid substitutions at E233, L234,L235, G236, G237, D265, D270, N297, E318, K320, K322, A327, A330, P331,or P329 (numbering according to the EU index of Kabat (Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991))).

In some embodiments, antibodies or conjugates comprising a non-native Fcregion described herein exhibit reduced or ablated binding to at leastone of Fcγ receptors CD16a, CD32a, CD32b, CD32c, and CD64 as compared toa polypeptide construct comprising a native Fc region. In some cases,the antibodies or conjugates described herein exhibit reduced or ablatedbinding to CD16a, CD32a, CD32b, CD32c, and CD64 Fcγ receptors.

CDC refers to a form of cytotoxicity in which the complement cascade isactivated by the complement component C1q binding to antibody Fc. Insome embodiments, antibodies or conjugates comprising a non-native Fcregion described herein exhibit at least a 5%, 10%, 15%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90% or greater reduction in C1q binding compared toan antibody or conjugate comprising a wild-type Fc region. In somecases, antibodies or conjugates comprising a non-native Fc region asdescribed herein exhibit reduced CDC as compared to antibodies orconjugates comprising a wild-type Fc region. In some embodiments,antibodies or conjugates comprising a non-native Fc region as describedherein exhibit at least a 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90% or greater reduction in CDC compared to antibodies orconjugates comprising a wild-type Fc region. In some cases, antibodiesor conjugates comprising a non-natural Fc variant as described hereinexhibit negligible CDC as compared to antibodies or conjugatescomprising a wild-type Fc region.

In some embodiments, the Fc variants herein are minimally glycosylatedor have reduced glycosylation relative to a wild-type sequence. In someembodiments, deglycosylation is accomplished with a mutation of N297A,or by mutating N297 to any amino acid which is not N. In someembodiments, deglycosylation is accomplished by disrupting the motifN-Xaa1-Xaa2-Xaa3 (SEQ ID NO:175), wherein N=asparagine; Xaa1=any aminoacid except P (proline); Xaa2=T (threonine), S (serine) or C (cysteine);and Xaa3=any amino acid except P (proline). In one embodiment, theN-Xaa1-Xaa2-Xaa3 (SEQ ID NO:175) motif refers to residues 297-300 asdesignated according to Kabat et al., 1991. In some embodiments, amutation to any one or more of N, Xaa1, Xaa2, or Xaa3 results indeglycosylation of the Fc variant.

In some embodiments, variants of antibody IgG constant regions (e.g., Fcvariants) possess a reduced capacity to specifically bind Fcγ receptorsor have a reduced capacity to induce phagocytosis. In some embodiments,variants of antibody IgG constant regions (e.g., Fc variants) possess areduced capacity to specifically bind Fcγ receptors and have a reducedcapacity to induce phagocytosis. For example, in some embodiments, an Fedomain is mutated to lack effector functions, typical of a “dead” Fcdomain. For example, in some embodiments, an Fc domain includes specificamino acid substitutions that are known to minimize the interactionbetween the Fc domain and an Fcγ receptor. In some embodiments, an Fcdomain monomer is from an IgG1 antibody and includes one or more ofamino acid substitutions L234A, L235A, G237A, and N297A (as designatedaccording to the EU numbering system per Kabat et al., 1991). In someembodiments, one or more additional mutations are included in such IgG1Fc variant. Non-limiting examples of such additional mutations for humanIgG1 Fc variants include E318A and K322A. In some instances, a humanIgG1 Fc variant has up to 12, 11, 10, 9, 8, 7, 6, 5 or 4 or fewermutations in total as compared to wild-type human IgG1 sequence. In someembodiments, one or more additional deletions are included in such IgG1Fc variant. For example, in some embodiments, the C-terminal lysine ofthe Fc IgG1 heavy chain constant region is deleted, for example toincrease the homogeneity of the polypeptide when the polypeptide isproduced in bacterial or mammalian cells. In some instances, a humanIgG1 Fc variant has up to 12, 11, 10, 9, 8, 7, 6, 5 or 4 or fewerdeletions in total as compared to wild-type human IgG1 sequence.

In some embodiments, an Fc domain monomer is from an IgG2 or IgG4antibody and includes amino acid substitutions A330S, P331S, or bothA330S and P331S. The aforementioned amino acid positions are definedaccording to Kabat, et al. (1991). The Kabat numbering of amino acidresidues can be determined for a given antibody by alignment at regionsof homology of the sequence of the antibody with a “standard” Kabatnumbered sequence. In some embodiments, the Fc variant comprises a humanIgG2 Fc sequence comprising one or more of A330S, P331S and N297A aminoacid substitutions (as designated according to the EU numbering systemper Kabat, et al. (1991). In some embodiments, one or more additionalmutations are included in such IgG2 Fc variants. Non-limiting examplesof such additional mutations for human IgG2 Fc variant include V234A,G237A, P238S, V309L and H268A (as designated according to the EUnumbering system per Kabat et al. (1991)). In some instances, a humanIgG2 Fc variant has up to 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or fewermutations in total as compared to wild-type human IgG2 sequence. In someembodiments, one or more additional deletions are included in such IgG2Fc variant. For example, in some embodiments, the C-terminal lysine ofthe Fc IgG2 heavy chain constant region is deleted, for example toincrease the homogeneity of the polypeptide when the polypeptide isproduced in bacterial or mammalian cells. In some instances, a humanIgG2 Fc variant has up to 12, 11, 10, 9, 8, 7, 6, 5 or 4 or fewerdeletions in total as compared to wild-type human IgG2 sequence.

When the Fc variant is an IgG4 Fc variant, in some embodiments, such Fcvariant comprises a S228P mutation (as designated according to Kabat, etal. (1991)), e.g., as represented in SEQ ID NO:104 in Table 7. In someinstances, a human IgG4 Fc variant has up to 12, 11, 10, 9, 8, 7, 6, 5,4, 3, 2 or 1 mutation(s) in total as compared to wild-type human IgG4sequence.

In some embodiments, the Fc variant includes at least one of themutations L234A, L235A, G237A or N297A of an IgG1 Fc region or at leastone of the mutations A330S, P331S or N297A of an IgG2 Fc region. In someembodiments, the Fc variant includes at least two of the mutationsL234A, L235A, G237A or N297A of an IgG1 Fc region or at least two of themutations A330S, P331S or N297A of an IgG2 Fc region. In someembodiments, the Fc variant includes at least three of the mutationsL234A, L235A, G237A or N297A of an IgG1 Fc region or consists of themutations A330S, P331S and N297A of an IgG2 Fc region. In someembodiments, the Fc variant consists of the mutations L234A, L235A,G237A and N297A.

In some embodiments, the Fc variant exhibits reduced binding to an Fcreceptor of the subject compared to the wild-type human IgG Fc region.In some embodiments, the Fc variant exhibits ablated binding to an Fcreceptor of the subject compared to the wild-type human IgG Fc region.In some embodiments, the Fc variant exhibits a reduction of phagocytosiscompared to the wild-type human IgG Fc region. In some embodiments, theFc variant exhibits ablated phagocytosis compared to the wild-type humanIgG Fc region.

Antibody-dependent cell-mediated cytotoxicity, which is also referred toherein as ADCC, refers to a form of cytotoxicity in which secreted Igbound onto Fc receptors (FcRs) present on certain cytotoxic cells (e.g.,Natural Killer (NK) cells and neutrophils) enabling these cytotoxiceffector cells to bind specifically to an antigen-bearing target celland subsequently kill the target cell. Antibody-dependent cell-mediatedphagocytosis, which is also referred to herein as ADCP, refers to a formof cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs)present on certain phagocytic cells (e.g., macrophages) enabling thesephagocytic effector cells to bind specifically to an antigen-bearingtarget cell and subsequently engulf and digest the target cell.Ligand-specific high-affinity IgG antibodies directed to the surface oftarget cells can stimulate the cytotoxic or phagocytic cells and can beused for such killing. In some embodiments, antibodies or conjugatescomprising an Fc variant as described herein exhibit reduced ADCC orADCP as compared to antibodies or conjugates comprising a wild-type Fcregion. In some embodiments, antibodies or conjugates comprising an Fcvariant as described herein exhibit at least a 5%, 10%, 15%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90% or greater reduction in ADCC or ADCPcompared to antibodies or conjugates comprising a wild-type Fc region.In some embodiments, antibodies or conjugates comprising an Fc variantas described herein exhibit ablated ADCC or ADCP as compared toantibodies or conjugates comprising a wild-type Fc region.

Complement-directed cytotoxicity, which is also referred to herein asCDC, refers to a form of cytotoxicity in which the complement cascade isactivated by the complement component C1q binding to antibody Fc. Insome embodiments, antibodies or conjugates comprising an Fc variant asdescribed herein exhibit at least a 5%, 10%, 15%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90% or greater reduction in C1q binding compared toantibodies or conjugates comprising a wild-type Fc region. In somecases, antibodies or conjugates comprising an Fc variant as describedherein exhibit reduced CDC as compared to antibodies or conjugatescomprising a wild-type Fc region. In some embodiments, antibodies orconjugates comprising an Fc variant as described herein exhibit at leasta 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greaterreduction in CDC compared to antibodies or conjugates comprising awild-type Fc region. In some cases, antibodies or conjugates comprisingan Fc variant as described herein exhibit negligible CDC as compared toantibodies or conjugates comprising a wild-type Fc region.

Fc variants herein include those that exhibit reduced binding to an Fcγreceptor compared to the wild-type human IgG Fc region. For example, insome embodiments, an Fc variant exhibits binding to an Fcγ receptor thatis less than the binding exhibited by a wild-type human IgG Fc region toan Fcγ receptor, as described in the Examples. In some instances, an Fcvariant has reduced binding to an Fcγ receptor by a factor of 10%, 20%30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100%(fully ablated effector function). In some embodiments, the reducedbinding is for any one or more Fcγ receptor, e.g., CD16a, CD32a, CD32b,CD32c, or CD64.

In some instances, the Fc variants disclosed herein exhibit a reductionof phagocytosis compared to its wild-type human IgG Fc region. Such Fcvariants exhibit a reduction in phagocytosis compared to its wild-typehuman IgG Fc region, wherein the reduction of phagocytosis activity ise.g., by a factor of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,96%, 97%, 98%, 99% or 100%. In some instances, an Fc variant exhibitsablated phagocytosis compared to its wild-type human IgG Fc region.

In some embodiments, a Q-tag of the present disclosure is attached to anantibody, polypeptide, small molecule (e.g., a small molecule agonist orantagonist), natural product, DNA molecule, RNA molecule (e.g., RNA,siRNA, antisense oligonucleotide, CRISPR guide RNA, etc.), other nucleicacid, CRISPR complex, or carbohydrate.

In some embodiments, the Q-tag is attached to the heavy chain of theantibody. In some embodiments, the Q-tag is attached to the heavy chainof the antibody via a linker (e.g., an amino acid or other chemicallinker). In some embodiments, the Q-tag is attached to the heavy chainof the antibody (e.g., fused in frame with the heavy chain). In someembodiments, the Q-tag is attached at the C-terminus of the heavy chainof the antibody. In some embodiments, the Q-tag is fused to theC-terminus of the heavy chain of the antibody (e.g., in frame andcontiguous with the amino acid sequence of the C-terminus). In someembodiments, the Q-tag is within the Fc domain of the antibody. In someembodiments, the Q tag is naturally occurring. For example, mutation ofN297 to N297A exposes Q295 of the antibody, where the conjugation couldoccur. In certain embodiments wherein the Fc region comprises an N297Asubstitution, the conjugate further comprises an immunomodulatingoligonucleotide P attached to the Q295 residue as shown in the followingformula

wherein L is a linker moiety connected to Q295 via an amide bond.

In some embodiments, the Q-tag comprises one or more sequences shown inTable 3.

TABLE 3 Q-tag Peplide Sequences SEQ ID NO. Peptide Sequences 39LSLSPGLLQGG-OH 40 WPAQGPT 41 WPQGPT 42 WAPQGPT 43 WAQGPT 44 TPGQAPW 45PNPQLPF 46 RPQQF 47 RPQGF 48 RPQGFPP 49 RPQGFGPP 50 RPRPQQF 51 LSQSKVLG52 WGGQLL 53 WALQRPHYSYPD 54 WALQRPYTLTES 55 WALQGPYTLTES

In some embodiments, the conjugate provided herein is to a targetspecific cell and tissue in a body for targeted delivery of a conjugatedpayload polynucleotide. In certain embodiments, the cell targeted by theconjugate provided herein is a natural killer cell. In certainembodiments, the cell targeted by the conjugate provided herein ismyeloid cell. In certain embodiments, the cell targeted by the conjugateprovided herein is B cell or T cell. In certain embodiments, the celltargeted by the conjugate provided herein is a neutrophil. In certainembodiments, the cell targeted by the conjugate provided herein is amonocyte. In certain embodiments, the cell targeted by the conjugateprovided herein is a macrophage. In certain embodiments, the celltargeted by the conjugate provided herein is a dendritic cell (DC). Incertain embodiments, the cell targeted by the conjugate provided hereinis a mast cell. In certain embodiments, the cell targeted by theconjugate provided herein is a tumor-associated macrophage (TAM). Incertain embodiments, the cell targeted by the conjugate provided hereinis a myeloid-derived suppressor cell (MDSC).

In some embodiments, an antibody or conjugate of the present disclosurecan be delivered as a naked protein-drug conjugate, or as a protein-drugconjugate formulated with a carrier and delivered, e.g., as encapsulatedor as part of a nanocarrier, nanoparticle, liposome, polymer vesicle, orviral envelope. In some embodiments, an antibody or conjugate of thepresent disclosure can be delivered intracellularly, e.g., byconjugation to a protein-transduction domain or mimic. In someembodiments, an antibody or conjugate of the present disclosure can bedelivered by electroporation or microinjection.

In some embodiments, a conjugate of the present disclosure targets morethan one population or type of cell, e.g., from those described supra.In some embodiments, a conjugate of the present disclosure targets bothB-cells and monocytes. In some embodiments, a conjugate of the presentdisclosure targets both B-cells, monocytes and/or DCs. In someembodiments, a conjugate of the present disclosure targets both NKs andDCs.

In certain embodiments, the antigen-binding moiety in the conjugateprovided herein is an antibody or an antigen-binding fragment thereof(e.g., F(ab)₂ or Fab) or an engineered derivative thereof (e.g., Fcab ora fusion protein (e.g., scFv)). In certain embodiments, theantigen-binding moiety in the conjugate provided herein is a human orchimeric (e.g., humanized) antibody.

In some embodiments, the antibodies or conjugates target one or moretype(s) of normal cell selected from T cells, B cells, natural killercells, neutrophils, mast cells, macrophages, antigen-presenting cells(APC), basophils, and eosinophils. In some embodiments, the antibodiesor conjugates target a normal APC. In some embodiments, the antibodiesor conjugates target one or more type(s) of normal APC selected from Bcells, monocytes, dendritic cells, Langerhans cells, keratinocytes,endothelial cells, astrocytes, fibroblasts, and oligodendrocytes. Insome embodiments, the antibodies or conjugates target a normal B cell.In some embodiments, the antibodies or conjugates target a normaldendritic cell. In some embodiments, the antibodies or conjugates targeta normal macrophage. In some embodiments the antibodies or conjugatestargeting one or more type(s) of normal cells do not target an abnormalcell, such as a cancer cell.

In some embodiments, an antibody or conjugate of the present disclosurecan comprise a multispecific (e.g., bispecific) antibody. For example,in some embodiments, an antibody of the present disclosure is abispecific antibody comprising 2 antigen binding domains that binddifferent targets expressed on B cells. In some embodiments, an antibodyof the present disclosure is a bispecific antibody comprising an antigenbinding site that binds a target expressed on a B cell and an antigenbinding site that binds a target expressed on another cell (e.g., amonocyte). In some embodiments, an antibody of the present disclosure isa bispecific antibody comprising an antigen binding site that binds atarget expressed on an immune cell and an antigen binding site thatbinds a target expressed on the surface of a cancer cell.

In certain embodiments, the antibody binds to an antigen expressed by aB cell. Exemplary antigens expressed by B cells that can be targeted bythe conjugates provided herein include, but are not limited to,B220/CD45R, B7-1/CD80, B7-2/CD86, BCMA/TNFRSF17, BLIMP1/PRDM1, C1qR1/CD93, CD117/c-kit, CD11b/Integrin alpha M, CD19, CD1c/BDCA-1, CD1d,CD20, CD21, CD23/Fc epsilon RII, CD24, CD25/IL-2 R alpha, CD27/TNFRSF7,CD34, CD37, CD38, CD40/TNFRSF5, CD43, CD5, CD69, CD72, CD83, CXCR4,CXCR5, DEP-1/CD148, EMMPRIN/CD147, FCRL3/FcRH3, Flt-3/Flk-2, HLA-DR,IgM, IL-10, IL-12 R beta 2, IL-12/IL-35 p35, IL-21, IL-21 R, IL-27 Ralpha/WSX-1/TCCR, IL-27/IL-35 EBI3 Subunit, IL-3 R alpha/CD123, IL-4 Ralpha, IL-6 receptor, IL-7 R alpha/CD127, IRF4, MHC class II (I-A/I-E),Neprilysin/CD10, Pax5/BSAP, Sca-1/Ly6, Siglec-2/CD22, STAT1, STAT3,Syndecan-1/CD138, TACI/TNFRSF13B, TGF-beta, TIM-1/KIM-1/HAVCR, TLR4. Inparticular embodiments, B cell specific antigens are selected from CD1,CD2, CD5, CD9, CD11, CD17, CD18, CD19, CD20, CD21/CD35, CD22, CD23,CD24, CD25, CD27, CD30, CD38, CD40, CD45R/B220, CD69, CD70, CD78, CD79a(Igα), CD79b (Igβ), CD80, CD86, CD93 (C1Rqp), CD137/4-1BB, CD138, CD180,CD252/OX40L, CD267, CD268/BAFF-R, CD279/PD1, CD319, PDL-2, Pax-5, IgD,IgM, Notch 2, and TLR4.

In certain embodiments, the antibody binds to an antigen expressed by aT cell (e.g., on the surface of a T cell). Exemplary antigens expressedby a T-cell that can be targeted by the conjugates provided hereininclude, but are not limited to, T-cell costimulatory molecules, OX40,CD2, CD27, CDS, ICAM-1, LFA-1/CD11a/CD18, ICOS/CD278, 4-1BB/CD137, GITR,CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3,CD83, BLIMP1/PRDNM1, SIRPγ, TNFR2, B7-H4, A2AR, STING and TIGIT.

In certain embodiments, the antibody binds to an antigen expressed by adendritic cell. Exemplary antigens expressed by a dendritic cell thatcan be targeted by the conjugates provided herein include, but are notlimited to, B220/CD45R, BATF3, BST-2/Tetherin, CD11b/Integrin alpha M,CD11c, CD14, CD163, CD19, CD1c/BDCA-1, CD1d1, CD20, CD3, CD4, CD8,CLEC9a, CX3CR1, DC-SIGN/CD209, DEC-205/CD205, DLEC/CLEC4C/BDCA-2,E-Cadherin, EpCAM/TROP1, F4/80, Fc epsilon RI alpha, Fc gamma RI/CD64,Fc gamma RIA/CD64, Fc gamma RIB/CD64, Fc gamma RIII (CD16), Fc gammaRIIIA/CD16a, Fc gamma RIIIB/CD16b, FLT3, GFI-1, HLA-DR, IFN-alpha,IFN-beta, IFN-gamma, IGSF4A/SynCAM1, A2AR, Ikaros, IL-1 beta/IL-1F2,IL-10, IL-12, IL-2, IL-23, IL-3 R alpha/CD123, IL-6, iNOS, Integrinalpha E/CD103, IRF4, IRF8, Langerin/CD207, Ly-6G (Gr-1), Ly-6G/Ly-6C(Gr-1), MHC class II (I-A/I-E), MMR/CD206, NCAM-1/CD56, Neuropilin-1,NFIL3/E4BP4, Nitric Oxide, PU.1/Spi-1, SIRP alpha/CD172a, Spi-B,Thrombomodulin/BDCA-3, TLR7, TLR9, TNF-alpha, TREM2, and XCR1. Inparticular embodiments, dendritic cell specific antigens are selectedfrom CD1a, CD1b/c, CD4, CD8, CD11b, CD11c, CD40, CD45R/B220, CD49d,CD80, CD83, CD85a, CD85f, CD85g/ILT7, CD85i, CD85j, CD86, CD123, CD180,CD197/CCR7, CD205, CD206, CD207, CD208, CD209, CD273/B7-DC/PD-L2,CD303/BDCA-2, CD304/neuropilin-1, DC marker/33D1, F4/80, MHC class I,fascin, HLA-DR, STING, and Siglec H. In particular embodiments,dendritic cell specific antigens are plasmacytoid dendritic cellantigens selected from CD1a, CD1b, CD1c, CD4, CD8, CD11b, CD11c, CD40,CD45R/B220, CD49d, CD80, CD83, CD85g/ILT7, CD86, CD123, CD197 (CCR7),CD273 (B7-DC, PD-L2), CD303 (BDCA-2), CD304 (Neuropilin-1), DC Marker(33D1), F4/80, HLA-DR, MHC Class II, and Siglec H.

In certain embodiments, the antibody binds to an antigen expressed by amacrophage. Exemplary antigens expressed by a macrophage that can betargeted by the conjugates provided herein include, but are not limitedto, Activin A, AIF-1/Iba1, Arginase 1/ARG1, A2AR, B7-1/CD80, B7-2/CD86,Calcitonin R, CCL1/I-309/TCA-3, CCL11/Eotaxin, CCL14/HCC-1/HCC-3,CCL15/MIP-1 delta, CCL16/HCC-4, CCL17/TARC, CCL18/PARC, CCL19/MIP-3beta, CCL2/JE/MCP-1, CCL20/MIP-3 alpha, CCL22/MDC, CCL23/Ck beta 8-1,CCL23/MPIF-1, CCL24/Eotaxin-2/MPIF-2, CCL26/Eotaxin-3, CCL3/CCL4,CCL3/MIP-1 alpha, CCL4/MIP-1 beta, CCL5/RANTES, CCL8/MCP-2, CCR2, CCR5,CD11b/Integrin alpha M, CD11c, CD15/Lewis X, CD163, CD200 R1, CD200R1L,CD36/SR-B3, CD43, CD45, CD68/SR-D1, CLEC10A/CD301, COX-2,CX3CL1/Fractalkine, CX3CR1, CXCL1/GRO alpha/KC/CINC-1,CXCL10/IP-10/CRG-2, CXCL11/I-TAC, CXCL13/BLC/BCA-1, CXCL16, CXCL2/GRObeta/MIP-2/CINC-3, CXCL3/GRO gamma/CINC-2/DCIP-1, CXCL5/ENA-70,CXCL5/ENA-74, CXCL5/ENA-78, CXCL9/MIG, CXCR1/IL-8 RA, CXCR2/IL-8 RB,DC-SIGN/CD209, DEC-205/CD205, Dectin-1/CLEC7A, Dectin-2/CLEC6A, EMR1,F4/80, Fc epsilon RI alpha, Fc gamma RI/CD64, Fc gamma RIA/CD64, Fcgamma RIB/CD64, Fc gamma RII/CD32, Fc gamma RIII (CD16), FIZZ1/RELMalpha, Galectin-3, GATA-6, G-CSF, GITR Ligand/TNFSF18, GM-CSF, HLA-DR,ID2, IFN-gamma, IFN-gamma R1/CD119, IL-1 beta/IL-1F2, IL-1 RII, IL-10,IL-15, IL-17/IL-17A, IL-18/IL-1F4, IL-1ra/IL-1F3, IL-23, IL-4 R alpha,IL-6, IL-8/CXCL8, iNOS, Integrin alpha L/CD11a, IRF4, IRF5,LAMP-2/CD107b, Langerin/CD207, LILRB4/CD85k/ILT3, L-Selectin/CD62L, LXRalpha/NR1H3, Ly-6G (Gr-1), Ly-6G/Ly-6C (Gr-1), MARCO, M-CSF R/CD115,Mer, MERTK, MFG-E8, MHC class II (I-A/I-E), MMR/CD206, NFATC1, NGFI-Balpha/Nur77/NR4A1, PPAR delta/NR1C2, PPAR gamma/NR1C3, RANK/TNFRSF11A,RUNX3/CBFA3, Siglec-1/CD169, Siglec-3/CD33, Siglec-F, SIGNR1/CD209b,SIRP alpha/CD172a, SLAM/CD150, SOCS-3, Sphingosine Kinase 1/SPHK1,Sphingosine Kinase 2/SPHK2, SR-AI/MSR, SR-BI, STAT1, STAT6, STING,TGF-beta, TIM-4, TLR1, TLR2, TLR4, TLR8, TNF-alpha, TRACP/PAP/ACP5,TREM1, VCAM-1/CD106, VEGF, and YM1/Chitinase 3-like 3. In particularembodiments, macrophage specific antigens are selected from CD11a,CD11b, CD11c, CD14, CD15 (SSEA-1), CD16/32, CD33, CD64, CD68, CD80,CD85k (ILT3), CD86, CD105 (Endoglin), CD107b, CD115, CD163, CD195(CCR5), CD282 (TLR2), CD284 (TLR4), F4/80, GITRL, HLA-DR, Mac-2(Galectin-3), and MHC Class II.

In certain embodiments, the antibody binds to an antigen expressed by anNK cell. Exemplary antigens expressed by a NK cell that can be targetedby the conjugates provided herein include, but are not limited to,CD11b, CD11c, CD16/32, CD49b, CD56 (NCAM), CD57, CD69, CD94, CD122,CD158 (Kir), CD161 (NK-1.1), CD180, CD244 (2B4), CD314 (NKG2D), CD319(CRACC), CD328 (Siglec-7), CD335 (NKp46), A2AR, Ly49, Ly108, Va24-J18TCR (iNKT), granulysin, granzyme, perforin, SIRP-α, LAIR1, SIGLEC-3(CD33), SIGLEC-7, SIGLEC-9, LIR1 (ILT2, LILRB1), NKR-P1A (KLRB1),CD94-NKG2A, KLRG1, KIR2DL5A, KIR2DL5B, KIR2DL1, KIR2DL2, KIR2DL3,KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DS1, KIR2DS1, CD94-NKG2C/E,NKG2D, CD160 (BY55), CD16 (FcγRIIIA), NKp46 (NCR1), NKp30 (NCR3), NKp44(NCR2), DNAM1(CD226), CRTAM, CD2, CD7, CD11a, CD18, CD25, CD27, CD28,NTB-A (SLAMF6), PSGL1, CD96 (Tactile), CD100 (SEMA4D), NKp80 (KLRF1,CLEC5C), SLAMF7 (CRACC, CS1, CD319), STING, and CD244 (2B4, SLAMF4).

In certain embodiments, the antibody binds to an antigen expressed by amyeloid cell. Exemplary antigens expressed by a myeloid cell and can betargeted by the conjugated provided herein include, but are not limitedto, siglec-3, siglec 7, siglec 9, siglec 10, siglec 15, CD200, CD200R,LILRB1, LILRB2, LILRB3, LILRB4, LILRB5, M-CSF, CSF-1R, GM-CSF R, IL4 R,arginase, IDO, TDO, MPO, EP2, COX-2, CCR2, CCR-7, CXCR1, CX3CR1, CXCR2,CXCR3, CXCR4, CXCR7, c-Kit, CD244, L-selectin/CD62L, CD11b, CD11c, CD68,CD163, CD180, CD204, DEC205, IL-1R, CD31, SIRPα, SIRPβ, PD-L1,CEACAM-8/CD66b, CD103, BDCA-1, BDCA2. BDCA-4, CD123, STING, and TLT-7.

In certain embodiments, the antibody binds to an antigen expressed by anMDSC. Exemplary antigens expressed by an MDSC and can be targeted by theconjugated provided herein include, but are not limited to, siglec-3,Siglec 7, siglec 9, siglec 10, siglec 15, CD200, CD200R, LILRB1, LILRB2,LILRB3, LILRB4, LILRB5, M-CSF, CSF-1R, GM-CSF R, IL4 R, arginase, IDO,TDO, MPO, EP2, COX-2, CCR2, CCR-7, CXCR1, CX3CR1, CXCR2, CXCR3, CXCR4,CXCR7, c-Kit, CD244, L-selectin/CD62L, CD11b, CD11c, CD68, CD163, CD180,CD204, DEC205, IL-1R, CD31, SIRPα, SIRPβ, PD-L1, CEACAM-8/CD66b, CD103,BDCA-1, BDCA2. BDCA-4, CD123, and TLT-7.

In certain embodiments, the antibody binds to an antigen expressed by aTAM. Exemplary antigens expressed by a TAM and can be targeted by theconjugated provided herein include, but are not limited to, siglec-3,Siglec 7, siglec 9, siglec 10, siglec 15, CD200, CD200R, nerophilin 2(NRP2), B7-H3, B7-H4, LILRB1, LILRB2, LILRB3, LILRB4, LILRB5, M-CSF,CSF-1R, GM-CSF R, IL4 R, arginase, IDO, TDO, MPO, EP2, COX-2, CCR2,CCR-7, CXCR1, CX3CR1, CXCR2, CXCR3, CXCR4, CXCR7, c-Kit, CD244,L-selectin/CD62L, CD11b, CD11c, CD68, CD163, CD204, DEC205, IL-1R, CD31,MARCO, TREM2, CD81, APOE, SIRPα, SIRPβ, PD-L1, CEACAM-8/CD66b, CD103,BDCA-1, BDCA2. BDCA-4, CD123, and ILT-7.

In certain embodiments, the antibody binds to an antigen specific to aNK cell. In certain embodiments, an NK cell is targeted by an anti-CD56antibody. In certain embodiments, the antibody is an anti-CD56 antibody.In certain embodiments, the antibody is a monoclonal anti-CD56 antibody.In certain embodiments, the antibody is a murine anti-CD56 antibody. Incertain embodiments, the murine anti-CD56 antibody is clone 5.1H11(BioLegend, Cat No: 362502). In certain embodiments, the murineanti-CD56 antibody is clone MEM-188 (BioLegend, 304601). In certainembodiments, the murine anti-CD56 antibody is clone QA17A16 (BioLegend,Cat No: 392402). In certain embodiments, the antibody is a humanizedanti-CD56 antibody. In certain embodiments, the antibody is a humananti-CD56 antibody. In certain embodiments, the antibody is a humanizedanti-CD56 antibody

B cells can be targeted by anti-CD38, anti-CD79b, anti-CD30, anti-CD22,or anti-CD20, anti-CD19 antibodies or antigen-binding fragments thereofor engineered derivatives thereof. Plasmacytoid dendritic cells (pDCs)can be targeted by anti-DEC205, anti-CD304 (BDCA4), anti-CD303 (BDCA2),anti-CD40, anti-CD74, or anti-CD123 antibodies or antigen-bindingfragments thereof or engineered derivatives thereof. Macrophages can betargeted by anti-CD163, anti-CD40, anti-CD74, anti-CD206, or anti-CD123antibodies or antigen-binding fragments thereof or engineeredderivatives thereof. In some embodiments, a conjugate of the presentdisclosure comprises an immunomodulating oligonucleotide as describedherein conjugated to a polypeptide, carbohydrate, or other compound thatassociates with or binds a target antigen described herein (e.g., CD22,a B cell antigen, a macrophage antigen, and so forth).

Non-limiting examples of anti-CD38 antibodies are daratumumab,SAR650984, MOR202, or any one of antibodies Ab79, Ab19, Ab43, Ab72, andAb110 disclosed in WO 2012/092616, the disclosure of these antibodies isincorporated herein by reference. A non-limiting example of ananti-CD79b antibody is huMA79b v28 disclosed in WO 2014/011521. Anon-limiting example of an anti-CD22 antibody is 10F4 disclosed in US2014/0127197. A non-limiting example of an anti-CD20 antibody isrituximab. A non-limiting example of an anti-DEC205 antibody is providedin US 2010/0098704, the antibodies of which are incorporated herein byreference. Non-limiting examples of anti-CD40 antibodies are lucatumumaband dacetuzumab. A non-limiting example of an anti-CD304 antibody isvesencumab.

In some embodiments, the antibody is selected from the group consistingof an anti-CD20 antibody, anti-CD22 antibody, anti-CD30 antibody, antiCD37 antibody, anti-CD38 antibody, anti-CD40 antibody, anti-CD74antibody, anti-CD79b antibody, anti-CD205 antibody, anti-CD274 antibody,anti-CD303 antibody, anti-CD304 antibody, anti-CD19 antibody, anti-CD1antibody, anti-CD2 antibody, anti-CD3 antibody, anti-CD5 antibody,anti-CD6 antibody, anti-CD9 antibody, anti-CD11 antibody, anti-CD18antibody, anti-CD21 antibody, anti-CD23 antibody, anti-CD24 antibody,anti-CD25 antibody, anti-CD26 antibody, anti-CD44 antibody, anti-CD45Rantibody, anti-CD49 antibody, anti-CD66 (Carcinoembrionic antigen, CEA)antibody, anti-CD93 antibody, anti-CD52 antibody, anti-CD56 antibody,anti-CD123 antibody, anti-CD138 antibody, anti-CD163 antibody,anti-SLAMF7 antibody, anti-CD180 antibody, anti-DEC205 antibody, andanti-CD206 antibody. In some embodiments, the antibody is an anti-CD20antibody. In some embodiments, the antibody is an anti-CD22 antibody.

In some embodiments, the CpG-Ab immunoconjugate specifically binds to atumor associated antigen of the cancer being treated by the presentmethod. Examples of tumor associated antigens (TAAs) that can betargeted by the CpG-Ab immunoconjugate of the present disclosureinclude, but are not limited to, sequences comprising all or part of thesequences of DLL3, GCC, GPA33, tissue factor (TF), TEM8, FOLR1, CEACAM5,LRRC15, Claudin18.2, AXL, CA9, CD155, AMHR2, NTSE, FLT1, nectin 4, 5T4MT-1/MMP-14, DKK1, myostatin, sema4D, Trop-2, HER3, Her2, Her2/neu,HER1, VWF, IGF-1, GRP78, CXCR4, cMET, vitmentin, VEGFR2, VEGFR1, VEGF,VEGF-A, TYRP1 (glycoprotein 75), TWEAK receptor, tumor antigenCTAA16.88, TRAIL-R2, TRAIL-R1, TNF-alpha, TGF-beta, TGF beta 2, TGF beta1, TFPI, tenascin C, TEM1, TAG-72, STEAPI, sphingosine-1-phosphate,SOST, SLAMF7, BCL-2, selectin P, SDC1, sclerostin, RTN4, RON, Rhesusfactor, RHD, respiratory syncytial virus, RANKL, rabies virusglycoprotein, PDGF-R beta, phosphatidylserine, phosphate-sodiumco-transporter, PDGF-R alpha, PDCD1, PD-1, PD-L1, PCSK9, oxLDL, OX-40,NRP1, Notch receptor 4, Notch receptor 3, Notch receptor 2, Notchreceptor 1, NOGO-A, NGF, neural apoptosis-regulated proteinase 1, NCA-90(granulocyte antigen), NARP-1, N-glycolylneuraminic acid, myostatin,myelin-associated glycoprotein, mucin CanAg, MSLN, MS4A1, MIF, MCP-1,LTA, LOXL2, lipoteichoic acid, LINGO-1, LFA-1 (CD11a), Lewis-Y antigen,L-selectin (CD62L), KIR, KIR ligand, ITGB2 (CD18), ITGA2, interferonreceptor, interferon gamma-induced protein, integrin αvβ, integrinαIIβ3, integrin α7β7, integrin α5β1, integrin α4β7, integrin α4,insulin-like growth factor I receptor, Influenza A hemagglutinin, ILGF2,IL9, IL6, IL4, IL3 IRA, IL23, ILI 7A, IL-6 receptor, IL-6, IL-S, IL-4,IL-23, IL-22, IL-I, IL-I 7A, IL-I 7, IL-13, IL-I 2, IL-I, IL 20, IGHE,IGF-I, IGF-I receptor, IgE Fc region, IFN-gamma, IFN-alpha, ICAM-1(CD54), human TNF, human scatter factor receptor kinase, Hsp90, HNGF,HLA-DR, HIV-1, histone complex, HHGFR, HGF, hepatitis B surface antigen,GUCY2C, GPNMB, GMCSF receptor alpha-chain, glypican 3, GD3 ganglioside,GD2, ganglioside GD2, Frizzled receptor, folate receptor 1, folatehydrolase, fibronectin extra domain-B, fibrin II, beta chain, FAP, Fprotein of respiratory syncytial virus, ERBB3, episialin, EpCAM,endotoxin, EGFR, EGFL7, E. coli shiga toxin type-2, E. coli shiga toxintype-I, DRS, DPP4, DLL4, dabigatran, cytomegalovirus glycoprotein B,CTLA-4, CSF2, CSF1R, clumping factor A, CLDN6, CLDN18.1, CLDN18.2,ch4DS, CFD, CEA-related antigen, CEA, CD80, CD79B, CD74, CD73, CD70,CD6, CD56, CD52, CD51, CD5, CD44 v6, CD41, CD40 ligand, CD40, CD47, CD4,CD39, CD38, CD37, CD33, CD30 (TNFRSF8), CD123, CD138, CD3 epsilon, CD3,CD28, CD274, CD27, CD2S (a chain of IL-2 receptor), CD23 (IgE receptor),CD221, CD22, CD200, CD20, CD2, CD19, CD137, CD142, CD154, CD152, CD15,CD147 (basigin), CD140a, CD125, CD11, CD-18, CCR5, CCR4, CCL11(eotaxin-I), cardiac myosin, carbonic anhydrase 9 (CA-IX), Canis lupusfamiliaris IL31, CA-125, C5, C242 antigen, C-X-C chemokine receptor type4, beta-amyloid, BAFF, B7-H3, B-lymphoma cell, AOC3 (VAP-I), anthraxtoxin, protective antigen, angiopoietin 3, angiopoietin 2,alpha-fetoprotein, AGS-22M6, adenocarcinoma antigen, ACVR2B, activinreceptor-like kinase I, 5T4, 5AC, 4-IBB, 1-40-beta-amyloid, EGFR,EGFRvIII, gp100 or Pmel17, CEA, MART-1/Melan-A, MAGE-A1, MAGE-A2,MAGE-A3, MAGE-A4, MUC-1, GPNMB, HMW-MAA, TIM1, ROR1, CD19, gp100,Dipeptidyl peptidase IV (DPPIV), adenosine deaminase-binding protein(ADAbp), cyclophilin b, Colorectal associated antigen(CRC)-C017-1A/GA733, Carcinoembryonic Antigen (CEA) and its immunogenicepitopes CAP-1 and CAP-2, etv6, amll, Prostate Specific Antigen (PSA)and its immunogenic epitopes PSA-1, PSA-2, and PSA-3, prostate-specificmembrane antigen (PSMA), T-cell receptor/CD3-zeta chain, MAGE-family oftumor antigens (e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5,MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12,MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1,MAGE-C2, MAGE-C3, MAGE-C4, MAGE-05), GAGE-family of tumor antigens(e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8,GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53,MUC family (e.g. MUC1, MUC16, etc; see e.g. U.S. Pat. No. 6,054,438;WO98/04727; or WO98/37095), p21ras, RCAS1, alpha-fetoprotein,E-cadherin, alpha-catenin, beta-catenin and gamma-catenin, p120ctn,PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC),fodrin, Connexin 37, Ig-idiotype, p15, gp75, GM2 and GD2 gangliosides,Smad family of tumor antigens brain glycogen phosphorylase, SSX-1, SSX-2(HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1 and CT-7, and c-erbB-2 andviral antigens such as the HPV-16 and HPV-18 E6 and E7 antigens and theEBV-encoded nuclear antigen (EBNA)-1, βhCG, WT1, TRP-2, NY-BR-1,NY-CO-58, MN (gp250), Telomerase, and germ cell derived tumor antigens.Tumor associated antigens also include the blood group antigens, forexample, Lea, Leb, LeX, LeY, H-2, B-1, B-2 antigens. Tumor associatedantigen can be identified using methods known in the art, such asdisclosed in Zhang et al. Supra.

Particularly, in some embodiments, the CpG-Ab immunoconjugatespecifically binds to a tumor associated antigen selected from CD19,CD20, CD22, CD25, CD30, CD33, CD38, CD40, CD44, CD45R (B220), CD49,CD52, CD56, CD70, CD74, CD79a, CD79b, CD93, CD123, CD138, CD163, CD205,CD206, CD274, CD303, and CD304, folate receptor alpha, folate receptorbeta, mesothelin, PSMA, Her-2, EGFR, CLDN18.2, 5T4, CD47, nectin 4,transferrin receptor, integrin, cripto, EphA2, AGS-5, AGS-16, CanAg,EpCAM, IL4 receptor, IL2 receptor, Lewis Y, GPNMB, DLL3, GCC, GPA33,tissue factor (TF), and Trop2. In some embodiments, the tumor associatedantigen is expressed by tumor cells. In some embodiments, the tumorassociated antigen is expressed by stromal cells, e.g., part of thetumor stroma.

Certain aspects of the present disclosure relate to CD22 and anti-CD22antibodies. In some embodiments, CD22 refers to human CD22, and theantibodies bind human CD22. CD22 is also known as Siglec-2, and CD22gene and polypeptide sequences (e.g., human gene and polypeptidesequences) are known in the art. See, e.g., NCBI Gene ID No. 933 andNCBI Ref. Seq. Accession No. NP_001172028. In certain embodiments, theanti-CD22 antibody is an antibody comprising a VH and VL as shown belowin Table 4. Anti-CD22 Antibody Sequences.

TABLE 4 Anti-CD22 Antibody Sequences SEQ ID Name Domain NO: SequenceRFB4 VH 56 EVQLVESGGGLVKPG GSLKLSCAASGFAFS IYDMSWVRQTPEKRLEWVAYISSGGGTTYY PDTVKGRFTISRDNA KNTLYLQMSSLKSED TAMYYCARHSGYGSSYGVLFAYWGQGTLVT VSS RFB4 VL 57 DIQMTQTTSSLSASL GDRVTISCRASQDISNYLNWYQQKPDGTVK LLIYYTSILHSGVPS RFSGSGSGTDYSLTI SNLEQEDFATYFCQQGNTLPWTFGGGTKLE IK epratuzumab VH 58 QVQLVQSGAEVKKPG SSVKVSCKASGYTFTSYWLHWVRQAPGQGL EWIGYINPRNDYTEY NQNFKDKATITADES TNTAYMELSSLRSEDTAFYFCARRDITTFY WGQGTTVTVSS epratuzumab VL 59 DIQLTQSPSSLSASVGDRVTMSCKSSQSVL YSANHKNYLAWYQQK PGKAPKLLIYWASTR ESGVPSRFSGSGSGTDFTLTISSLQPEDIA TYYCHQYLSSVVTFG GGTKLEIK m971 VH 60 QVQLQQSGPGLVKPSQTLSLTCAISGDSVS SNSAAWNWIRQSPSR GLEWLGRTYYRSKWY NDYAVSVKSRITINPDTSKNQFSLQLNSVT PEDTAVYYCAREVTG DLEDAFDIWGQGTMV TVSS m971 VL 61DIQMTQSPSSLSASV GDRVTITCRASQTIW SYLNWYQQRPGKAPN LLIYAASSLQSGVPSRFSGRGSGTDFTLTI SSLQAEDFATYYCQQ SYSIPQTFGQGTKLE IK 10F4 VH 62EVQLVESGGGLVQPG GSLRLSCAASGYEFS RSWMNWVRQAPGKGL EWVGRIYPGDGDTNYSGKFKGRFTISADTS KNTAYLQMNSLRAED TAVYYCARDGSSWDW YFDVWGQGTLVTVSSEVQLVESGGGLVQPG 10F4 VL 63 MDIQMTQSPSSLSAS VGDRVTITCRSSQSIVHSVGNTFLEWYQQK PGKAPKLLIYKVSNR FSGVPSRFSGSGSGT DFTLTISSLQPEDFATYYCFQGSQFPYTFG QGTKVEIK RH1 VH 64 EVQLVESGGGLVQPG GSLRLSCAASGFAFSIYDMSWVRQAPGKGL EWVAYISSGGGTTYY PDTVKGRFTISRDNS KNTLYLQMNSLRAEDTAVYYCARHSGYGTH WGVLFAYWGRGTLVT VSS RH2 VH 65 QVQLLESGGGVVQPGGSLRLSCAASGFAFS IYDMNWVRQAPGKGL EWVSAISSGGGTTYY ADSVKGRFTISRDNAKNSLYLQMNSLRAED TAVYYCARHSGYGTH WGVLFAYWGRGTLVT VSS RH3 VH 66EVQLVESGGGLVQPG GSLRLSCAASGFTFS SYEMNWVRQAPGKGL EWVSYISSSGSTIYYADSVKGRFTISRDNA KNSLYLQMNSLRAED TAVYYCARHSGYGTH WGVLFAYWGRGTLVT VSS RH4VH 67 QVQLQESGPGLVKPS DTLSLTCTVSGFAFS IYDMSWIRQPPGKGL EWIAYISSGGGTTYYNPSLKSRVTISVDTS KNQFSLKLSSVTAAD TAVYYCARHSGYGTH WGVLFAYWGRGTLVT VSS RL1VL 68 DIQMTQSPSSLSASV GDRVTITCRASQDIH GYLNWYQQKPGKAPK LLIYYTSILHSGVPSRFSGSGSGTDFTLTI SSLQPEDFATYFCQQ GNTLPWTFGQGTKLE IK RL2 VL 69DIQMTQSPSSVSASV GDRVTITCRASQDIH GYLAWYQQKPGKAPK LLIYYTSSLQSGVPSRFSGSGSGTDFTLTI SSLQPEDFATYYCQQ GNTLPWTFGQGTKLE IK RL3 VL 70DIQMTQSPSSLSASV GDRVTITCRASQSIS SYLNWYQQKPGKAPK LLIYAASSLQSGVPSRFSGSGSGTDFTLTI SSLQPEDFATYYCQQ GNTLPWTFGQGTKLE IK RL4 VL 71EIVLTQSPATLSLSP GERATLSCRASQDIH GYLNWYQQKPGQAPR LLIYYTSILHSGIPARFSGSGPGTDFTLTI SSLEPEDFAVYYCQQ GNTLPWTFGGGTKLE IK RL5 VL 72DIVMTQTPLSLSVTP GQPASISCRASQDIH GYLNWYQQKPGQSPQ LLIYYTSILHSGVPDRFSGSGSGTDFTLKI SRVEAEDVGVYFCQQ GNTLPWTFGGGTKLE IK

TABLE 5 Anti-CD22 Antibody VH Sequences. Name Sequence SEQ ID NO RH1EVQLVESGGGLVQPGGSLRLSCAASGFAFSIYDMSWVRQAPGKGLEWVAYISSGG 64GTTYYPDTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHSGYGTHWGVLFA YWGRGTLVTVSSRH2 QVQLLESGGGVVQPGGSLRLSCAASGFAFSIYDMNWVRQAPGKGLEWVSAISSGG 65GTTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHSGYGTHWGVLFA YWGRGTLVTVSSRH3 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYEMNWVRQAPGKGLEWVSYISSSGS 66TIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHSGYGTHWGVLFAY WGRGTLVTVSS RH4QVQLQESGPGLVKPSDTLSLTCTVSGFAFSIYDMSWIRQPPGKGLEWIAYISSGGGT 67TYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARHSGYGTHWGVLFAYW GRGTLVTVSS

TABLE 6 Anti-CD22 Antibody VL Sequences. SEQ ID Name Sequence NO RL1DIQMTQSPSSLSASVGDRVT 68 ITCRASQDIHGYLNWYQQKP GKAPKLLIYYTSILHSGVPSRFSGSGSGTDFTLTISSLQP EDFATYFCQQGNTLPWTFGQ GTKLEIK RL2DIQMTQSPSSVSASVGDRVT 69 ITCRASQDIHGYLAWYQQKP GKAPKLLIYYTSSLQSGVPSRFSGSGSGTDFTLTISSLQP EDFATYYCQQGNTLPWTFGQ GTKLEIK RL3DIQMTQSPSSLSASVGDRVT 70 ITCRASQSISSYLNWYQQKP GKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQP EDFATYYCQQGNTLPWTFGQ GTKLEIK RL4ELVLTQSPATLSLSPGERAT 71 LSCRASADIHGYLNWYQQKP GQAPRLLIYYTSILHSGIPARFSGSGPGTDFTLTISSLEP EDFAVYYCQQGNTLPWTFGG GTKLEIK RL5DIVMTQTPLSLSVTPGQPAS 72 ISCRASADIHGYLNWYQQKP GASPQLLIYYTSILHSGVPDRFSGSGSGTDFTLKISRVEA EDVGVYFCQQGNTLPWTFGG GTKLEIK RL1DIQMTQSPSSLSASVGDRVT 73 N92A ITCRASQDIHGYLNWYQQKP GKAPKLLIYYTSILHSGVPSRFSGSGSGTDFTLTISSLQP EDFATYFCQQGATLPWTFGQ GTKLEIK RL1DIQMTQSPSSLSASVGDRVT 74 N92C ITCRASQDIHGYLNWYQQKP GKAPKLLIYYTSILHSGVPSRFSGSGSGTDFTLTISSLQP EDFATYFCQQGCTLPWTFGQ GTKLEIK RL1DIAMTQSPSSLSASVGDRVT 75 N92D ITCRASQDIHGYLNWYQQKP GKAPKLLIYYTSILHSGVPSRFSGSGSGTDFTLTISSLQP EDFATYFCQQGDTLPWTFGQ GTKLEIK RL1DIQMTQSPSSLSASVGDRVT 76 N92E ITCRASQDIHGYLNWYQQKP GKAPKLLIYYTSILHSGVPSRFSGSGSGTDFTLTISSLQP EDFATYFCQQGETLPWTFGQ GTKLEIK RL1DIQMTQSPSSLSASVGDRVT 77 N92F ITCRASQDIHGYLNWYQQKP GKAPKLLIYYTSILHSGVPSRFSGSGSGTDFTLTISSLQP EDFATYFCQQGFTLPWTFGQ GTKLEIK RL1DIQMTQSPSSLSASVGDRVT 78 N92G ITCRASQDIHGYLNWYQQKP GKAPKLLIYYTSILHSGVPSRFSGSGSGTDFTLTISSLQP EDFATYFCQQGGTLPWTFGQ GTKLEIK RL1DIQMTQSPSSLSASVGDRVT 79 N92H ITCRASQDIHGYLNWYQQKP GKAPKLLIYYTSILHSGVPSRFSGSGSGTDFTLTISSLQP EDFATYFCQQGHTLPWTFGQ GTKLEIK RL1DIQMTQSPSSLSASVGDRVT 80 N92I ITCRASQDIHGYLNWYQQKP GKAPKLLIYYTSILHSGVPSRFSGSGSGTDFTLTISSLQP EDFATYFCQQGITLPWTFGQ GTKLEIK RL1DIQMTQSPSSLSASVGDRVT 81 N92K ITCRASQDIHGYLNWYQQKP GKAPKLLIYYTSILHSGVPSRFSGSGSGTDFTLTISSLQP EDFATYFCQQGKTLPWTFGQ GTKLEIK RL1DIQMTQSPSSLSASVGDRVT 82 N92L ITCRASQDIHGYLNWYAQKP GKAPKLLIYYTSILHSGVPSRFSGSGSGTDFTLTISSLQP EDFATYFCQQGLTLPWTFGQ GTKLEIK RL1DIQMTQSPSSLSASVGDRVT 83 N92M ITCRASQDIHGYLNWYQQKP GKAPKLLIYYTSILHSGVPSRFSGSGSGTDFTLTISSLQP EDFATYFCQQGMTLPWTFGQ GTKLEIK RL1DIQMTQSPSSLSASVGDRVT 84 N92P ITCRASQDIHGYLNWYQQKP GKAPKLLIYYTSILHSGVPSRFSGSGSGTDFTLTISSLQP EDFATYFCQQGPTLPWTFGQ GTKLEIK RL1DIQMTQSPSSLSASVGDRVT 85 N92Q ITCRASQDIHGYLNWYQQKP GKAPKLLIYYTSILHSGVPSRFSGSGSGTDFTLTISSLQP EDFATYFCQQGQTLPWTFGQ GTKLEIK RL1DIQMTQSPSSLSASVGDRVT 86 N92R ITCRASADIHGYLNWYQQKP GKAPKLLIYYTSILHSGVPSRFSGSGSGTDFTLTISSLQP EDFATYFCQQGRTLPWTFGQ GTKLEIK RL1DIQMTQSPSSLSASVGDRVT 87 N92S ITCRASQDIHGYLNWYQQKP GKAPKLLIYYTSILHSGVPSRFSGSGSGTDFTLTISSLQP EDFATYFCQQGSTLPWTFGQ GTKLEIK RL1DIQMTQSPSSLSASVGDRVT 88 N92T ITCRASQDIHGYLNWYQQKP GKAPKLLIYYTSILHSGVPSRFSGSGSGTDFTLTISSLQP EDFATYFCAAGTTLPWTFGA GTKLEIK RL1DIQMTQSPSSLSASVGDRVT 89 N92V ITCRASQDIHGYLNWYQQKP GKAPKLLIYYTSILHSGVPSRFSGSGSGTDFTLTISSLQP EDFATYFCQQGVTLPWTFGQ GTKLEIK RL1DIQMTQSPSSLSASVGDRVT 90 N92W ITCRASQDIHGYLNWYQQKP GKAPKLLIYYTSILHSGVPSRFSGSGSGTDFTLTISSLQP EDFATYFCQQGWTLPWTFGQ GTKLEIK RL1DIQMTQSPSSLSASVGDRVT 91 N92Y ITCRASQDIHGYLNWYQQKP GKAPKLLIYYTSILHSGVPSRFSGSGSGTDFTLTISSLQP EDFATYFCQQGYTLPWTFGQ GTKLEIK

TABLE 13 Anti-CD22 antibody sequences Heavy chain Light chainQVQLLESGGGWQPGG DIQMTQSPSSLSASV SLRLSCAASGFAFSI GDRVTITCRASQDIHYDMNWVRQAPGKGLE GYLNWYQQKPGKAPK WVSAISSGGGTTYYA LLIYYTSILHSGVPSDSVKGRFTISRDNAK RFSGSGSGTDFTLTI NSLYLQMNSLRAEDT SSLQPEDFATYFCQQAVYYCARHSGYGTHW GATLPWTFGQGTKLE GVLFAYWGRGTLVTV IKRTVAAPSVFIFPPSSASTKGPSVFPLAP SDEQLKSGTASVVCL SSKSTSGGTAALGCL LNNFYPREAKVQWKVVKDYFPEPVTVSWNS DNALQSGNSQESVTE GALTSGVHTFPAVLQ QDSKDSTYSLSSTLTSSGLYSLSSVVTVPS LSKADYEKHKVYACE SSLGTQTYICNVNHK VTHQGLSSPVTKSFNPSNTKVDKKVEPKSC RGEC DKTHTCPPCPAPELL (SEQ ID NO: 181) GGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVD GVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKG QPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSD GSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPG RPQGFGPP (SEQ ID NO: 179) QVQLLESGGGVVQPGDIQMTQSPSSLSASV GSLRLSCAASGFAFS GDRVTITCRASQDIH IYDMNWVRQAPGKGLGYLNWYQQKPGKAPK EWVSAISSGGGTTYY LLIYYTSILHSGVPS ADSVKGRFTISRDNARFSGSGSGTDFTLTI KNSLYLQMNSLRAED SSLQPEDFATYFCQQ TAVYYCARHSGYGTHGSTLPWTFGQGTKLE WGVLFAYWGRGTLVT IKRTVAAPSVFIFPP VSSASTKGPSVFPLASDEQLKSGTASWCLL PSSKSTSGGTAALGC NNFYPREAKVQWKVD LVKDYFPEPVTVSWNNALQSGNSQESVTEQ 5GALTSGVHTFPAVLQ DSKDSTYSLSSTLTL SSGLYSLSSVVTVPSSKADYEKHKVYACEV SSLGTQTYICNVNHK THQGLSSPVTKSFNR PSNTKVDKKVEPKSC GECDKTHTCPPCPAPEAA (SEQ ID NO: 182) GAPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQ YNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSR EEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKS RWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGRPQGFGPP (SEQ ID NO: 180)

In some embodiments, the anti-CD22 antibody or conjugate comprises aheavy chain comprising the sequence of SEQ ID NO:179 or 180, and a lightchain comprising the sequence of SEQ ID NO:181 or 182.

Certain aspects of the present disclosure relate to Her2 and anti-Her2antibodies. In some embodiments, Her2 refers to human Her2, and theantibodies bind human Her2. Her2 is also known as erb-b2 receptortyrosine kinase 2 (ERBB2), Neu, NGL, TKR1, CD340, and MLN19. Her2 geneand polypeptide sequences (e.g., human gene and polypeptide sequences)are known in the art. See, e.g., NCBI Gene ID No. 2064 and NCBI Ref.Seq. Accession No. NP_001005862. In some embodiments, the CpG-Abimmunoconjugate specifically binds to a Her2 polypeptide, e.g., a humanHer2 polypeptide. In some embodiments, the CpG-Ab immunoconjugatespecifically binds to an extracellular domain of a Her2 polypeptide,e.g., a human Her2 polypeptide. In some embodiments, the CpG-Abimmunoconjugate specifically binds to a cell (e.g., a tumor cell) thatexpresses a Her2 polypeptide, e.g., a human Her2 polypeptide, on itscell surface. Antibodies that bind a Her2 polypeptide, e.g., a humanHer2 polypeptide, are known in the art. In some embodiments, theantibody comprises a VH domain comprising 1, 2, or 3 CDR sequences fromthe VH domain of the anti-Her2 antibody trastuzumab and/or a VL domaincomprising 1, 2, or 3 CDR sequences from the VL domain of the anti-Her2antibody trastuzumab. In some embodiments, the antibody comprises a VHdomain comprising 1, 2, or 3 CDR sequences from a VH domain comprisingthe amino acid sequence ofEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGT LVTVSS (SEQ IDNO:168) and/or a VL domain comprising 1, 2, or 3 CDR sequences from a VLdomain comprising the amino acid sequence ofDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO:169). Insome embodiments, the antibody comprises the VH domain of the anti-Her2antibody trastuzumab and/or the VL domain of the anti-Her2 antibodytrastuzumab. In some embodiments, the antibody comprises a VH domaincomprising the amino acid sequence ofEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGT LVTVSS (SEQ IDNO:168) and/or a VL domain comprising the amino acid sequence of

(SEQ ID NO: 169) DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQ GTKVEIK.In some embodiments, the antibody comprises a VH domain comprising 1, 2,or 3 CDR sequences from the VH domain of the anti-Her2 antibodypertuzumab and/or a VL domain comprising 1, 2, or 3 CDR sequences fromthe VL domain of the anti-Her2 antibody pertuzumab. In some embodiments,the antibody comprises a VH domain comprising 1, 2, or 3 CDR sequencesfrom a VH domain comprising the amino acid sequence ofEVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLV TVSS (SEQ IDNO:170) and/or a VL domain comprising 1, 2, or 3 CDR sequences from a VLdomain comprising the amino acid sequence ofDIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIK (SEQ ID NO:171). In someembodiments, the antibody comprises the VH domain of the anti-Her2antibody pertuzumab and/or the VL domain of the anti-Her2 antibodypertuzumab. In some embodiments, the antibody comprises a VH domaincomprising the amino acid sequence ofEVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLV TVSS (SEQ IDNO:170) and/or a VL domain comprising the amino acid sequence ofDIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIK (SEQ ID NO:171). In someembodiments, the anti-Her2 antibody is trastuzumab. In some embodiments,the anti-Her2 antibody is pertuzumab. In some embodiments, the anti-Her2antibody has one or more effector functions, including withoutlimitation ADCC and/or ADCP. In some embodiments, the anti-Her2 antibodycomprises a human Fc region, e.g., a human IgG Fc region. In someembodiments, the anti-Her2 antibody comprises a wild-type human IgG1,IgG2, or IgG4 Fc region. In some embodiments, the anti-Her2 antibodycomprises the antibody constant domain sequence of SEQ ID NO:178.

TABLE 14 Anti-Her2 antibody sequences Heavy chain Light chainEVQLVESGGGLVQPGGSLRL DIQMTQSPSSLSASVGDRVT SCAASGFNIKDTYIHWVRQAITCRASQDVNTAVAWYQQKP PGKGLEWVARIYPTNGYTRY GKAPKLLIYSASFLYSGVPSADSVKGRFTISADTSKNTAY RFSGSRSGTDFTLTISSLQP LQMNSLRAEDTAVYYCSRWGEDFATYYCQQHYTTPPTFGQ GDGFYAMDYWGQGTLVTVSS GTKVEIKRTVAAPSVFIFPPASTKGPSVFPLAPCSRSTSE SDEQLKSGTASVVCLLNNFY STAALGCLVKDYFPEPVTVSPREAKVQWKVDNALQSGNSQ WNSGALTSGVHTFPAVLQSS ESVTEQDSKDSTYSLSSTLTGLYSLSSVVTVPSSSLGTKT LSKADYEKHKVYACEVTHQG YTCNVDHKPSNTKVDKRVESLSSPVTKSFNRGEC KYGPPCPPCPAPEFLGGPSV (SEQ ID NO: 185)FLFPPKPKDTLMISRTPEVT CVVVDVSQEDPEVQFNWYVD GVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDS DGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKS LSLSLGRPQGFGPP (SEQ ID NO: 183)EVQLVESGGGLVQPGGSLRL DIQMTQSPSSLSASVGDRVT SCAASGFNIKDTYIHWVRQAITCKASQDVSIGVAWYQQKP PGKGLEWVARIYPTNGYTRY GKAPKLLIYSASYRYTGVPSADSVKGRFTISADTSKNTAY RFSGSGSGTDFTLTISSLQP LQMNSLRAEDTAVYYCSRWGEDFATYYCQQYY1YPYTFGQ GDGFYAMDYWGQGTLVTVSS GTKVEIKRTVAAPSVFIFPPASTKGPSVFPLAPSSKSTSG SDEQLKSGTASVVCLLNNFY GTAALGCLVKDYFPEPVTVSPREAKVQWKVDNALQSGNSQ WNSGALTSGVHTFPAVLQSS ESVTEQDSKDSTYSLSSTLTGLYSLSSVVTVPSSSLGTQT LSKADYEKHKVYACEVTHQG YICNVNHKPSNTKVDKKVEPLSSPVTKSFNRGEC KSCDKTHTCPPCPAPELLGG (SEQ ID NO: 188)PSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGRPQGFGPP (SEQ ID NO: 184)EVQLVESGGGLVQPGGSLRL SCAASGFTFTDYTMDWVRQA PGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLY LQMNSLRAEDTAVYYCARNL GPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSES TAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTY TCNVDHKPSNTKVDKRVESK YGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSQEDPEVQFNWYVDG VEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKGLPSSIEKTISKAKG QPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSD GSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSL SLSLGRPQGFGPP (SEQ ID NO: 186) EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQA PGKGLEWVADVNPNSGGSIY NQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNL GPSFYFDYWGQGTLVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK SCDKTHTCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSREEM TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGRPQGFGPP (SEQ ID NO: 187)

In some embodiments, the anti-Her2 antibody or conjugate comprises aheavy chain comprising the sequence of SEQ ID NO:183, 184, 186, or 187;and a light chain comprising the sequence of SEQ ID NO:185 or 188.

In some embodiments, an antibody of the present disclosure (e.g., ananti-CD22 antibody or anti-Her2 antibody) comprises an antibody constantdomain. In some embodiments, an antibody of the present disclosure(e.g., an anti-CD22 antibody or anti-Her2 antibody) comprises anantibody heavy chain constant domain and/or antibody light chainconstant domain listed in Table 7. In some embodiments, an antibody ofthe present disclosure (e.g., an anti-CD22 antibody or anti-Her2antibody) comprises an antibody heavy chain constant domain selectedfrom the group consisting of SEQ ID Nos:92-107 and 178. In someembodiments, an antibody of the present disclosure (e.g., an anti-CD22antibody or anti-Her2 antibody) comprises an antibody heavy chainconstant domain with a Q-tag at the C-terminus of the Fc region, e.g.,as shown in SEQ ID No: 95 or 178. In some embodiments, an antibody ofthe present disclosure (e.g., an anti-CD22 antibody or anti-Her2antibody) comprises two antibody heavy chains, each with a constantdomain, wherein each of the two antibody heavy chains comprises a Q-tagat the C-terminus of the Fc region, e.g., as shown in SEQ ID No: 95 or178. In some embodiments, an antibody of the present disclosure (e.g.,an anti-CD22 antibody or anti-Her2 antibody) comprises two antibodyheavy chains, each with a constant domain, wherein only one of the twoantibody heavy chains comprises a Q-tag at the C-terminus of the Fcregion, e.g., as shown in SEQ ID No: 95 or 178. In some embodiments, anantibody of the present disclosure (e.g., an anti-CD22 antibody oranti-Her2 antibody) comprises an antibody light chain constant domainselected from the group consisting of SEQ ID Nos:108-110.

TABLE 7  Antibody constant domain sequences SEQ ID Name NO: SequenceIgG1 92 ASTKGPSVFPLAPSSKSTSG wildtype GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG IgG1_AAA_ 93ASTKGPSVFPLAPSSKSTSG N297A GTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG IgG1_AAA 94 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPEAAGA PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPGIgG1_AAA + 95 ASTKGPSVFPLAPSSKSTSG S-tag GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGA PSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPGRPQGFGPP IgG1_N297A96 ASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG IgG1_D265A 97 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPGIgG1_N297A/ 98 ASTKGPSVFPLAPSSKSTSG D265A GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTP EVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYA STYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG IgG2 99ASTKGPSVFPLAPCSRSTSE STAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQT YTCNVDHKPSNTKVDKTVER KCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVQFNWYVDG VEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKC KVSNKGLPAPIEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPMLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPG IgG2Da 100 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVER KCCVECPPCPAPPVAGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDG VEVHNAKTKPREEQFNSTFR VVSVLTWHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQ PREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDG SFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLS LSPGIgG2Da_ 101 ASTKGPSVFPLAPCSRSTSE N297A STAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSNFGTQT YTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVF LFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFR VVSVLTVVHQDWLNGKEYKC KVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSL SLSPG IgG2_ 102ASTKGPSVFPLAPCSRSTSE N297A STAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQT YTCNVDHKPSNTKVDKTVER KCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVQFNWYVDG VEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKC KVSNKGLPAPIEKTISKTKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPMLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPG IgG2Da_ 103 ASTKGPSVFPLAPCSRSTSE D265ASTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVER KCCVECPPCPAPPVAGPSVF LFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDG VEVHNAKTKPREEQFNSTFR VVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKG QPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSD GSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSL SLSPGIgG4_ 104 ASTKGPSVFPLAPCSRSTSE S228P STAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTKT YTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSV FLFPPKPKDTLMISRTPEVT CVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTY RWSVLTVLHQDWLNGKEYKC KVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKN QVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGN VFSCSVMHEALHNHYTQKSL SLSLG IgG4_S228P_ 105ASTKGPSVFPLAPCSRSTSE D265A STAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKT YTCNVDHKPSNTKVDKRVES KYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVT CVVVAVSQEDPEVQFNWYVD GVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDS DGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKS LSLSLG IgG4_S228P, 106 ASTKGPSVFPLAPCSRSTSE L235ESTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES KYGPPCPPCPAPEFEGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVD GVEVHNAKTKPREEQFNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSRLTVDKSRWQEG NVFSCSVMHEALHNHYTQKS LSLSLGIgG4_S228P, 107 ASTKGPSVFPLAPCSRSTSE N297A STAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTKT YTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSV FLFPPKPKDTLMISRTPEVT CVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTY RVVSVLTVLHQDWLNGKEYK CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTK NQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG NVFSCSVMHEALHNHYTQKS LSLSLG IgG1_wt + 178ASTKGPSVFPLAPSSKSTSG S-tag GTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGRPQGFGPP Human Kappa 108RTVAAPSVFIFPPSDEQLKS GTASVVCLLNNFYPREAKVQ WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTK SFNRGEC Human Lambda 109GQPKANPTVTLFPPSSEELQ IGLC1 ANKATLVCLISDFYPGAVTV AWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWK SHRSYSCQVTHEGSTVEKTV APTECS Human Lambda 110GQPKAAPSVTLFPPSSEELQ IGLC2 ANKATLVCLISDFYPGAVTV AWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWK SHRSYSCQVTHEGSTVEKTV APTECS

In some embodiments, an antibody or conjugate of the present disclosurecomprises a VH domain comprising 1, 2, or 3 CDRs of a single antibodyshown in Table 8. In some embodiments, an antibody or conjugate of thepresent disclosure comprises a VH domain comprising the 3 CDRs of asingle antibody shown in Table 8. In some embodiments, an antibody orconjugate of the present disclosure comprises a VL domain comprising 1,2, or 3 CDRs of a single antibody shown in Table 8. In some embodiments,an antibody or conjugate of the present disclosure comprises a VL domaincomprising the 3 CDRs of a single antibody shown in Table 8. In someembodiments, an antibody or conjugate of the present disclosurecomprises a VH domain comprising 1, 2, or 3 CDRs of a single antibodyshown in Table 8 and a VL domain comprising 1, 2, or 3 CDRs of a singleantibody shown in Table 8. In some embodiments, an antibody or conjugateof the present disclosure comprises a VH domain comprising the 3 CDRs ofa single antibody shown in Table 8 and a VL domain comprising the 3 CDRsof a single antibody shown in Table 8. In some embodiments, an antibodyor conjugate of the present disclosure comprises a VH domain comprisinga CDR-H1 comprising the sequence of SEQ ID NO:113, a CDR-H2 comprisingthe sequence of SEQ ID NO:115, and a CDR-H3 comprising the sequence ofSEQ ID NO:116. In some embodiments, an antibody or conjugate of thepresent disclosure comprises a VH domain comprising a CDR-H1 comprisingthe sequence of SEQ ID NO:114, a CDR-H2 comprising the sequence of SEQID NO:189, and a CDR-H3 comprising the sequence of SEQ ID NO:116. Insome embodiments, an antibody or conjugate of the present disclosurecomprises a VL domain comprising a CDR-L1 comprising the sequence of SEQID NO:117, a CDR-L2 comprising the sequence of SEQ ID NO:119, and aCDR-L3 comprising the sequence of SEQ ID NO:120. In some embodiments, anantibody or conjugate of the present disclosure comprises a VL domaincomprising a CDR-L1 comprising the sequence of SEQ ID NO:118, a CDR-L2comprising the sequence of SEQ ID NO:177, and a CDR-L3 comprising thesequence of SEQ ID NO:120

TABLE 8 Anti-CD22 antibody CDR sequences Antibody CDR-H1 CDR-H2 CDR-H3CDR-L1 CDR-L2 CDR-L3 RH1 GFAFSIYD ISSGGGTT ARHSGYGTHWGVLFAY — — —(SEQ ID (SEQ ID (SEQ ID NO: 116) NO: 113) NO: 115) RH2 GFAFSIYD ISSGGGTTARHSGYGTHWGVLFAY — — — (SEQ ID (SEQ ID (SEQ ID NO: 116) NO: 113)NO: 115) RH3 GFTFSSYE ISSSGSTI ARHSGYGTHWGVLFAY — — — (SEQ ID (SEQ ID(SEQ ID NO: 116) NO: 114) NO: 189) RH4 GFAFSIYD ISSGGGTTARHSGYGTHWGVLFAY — — — (SEQ ID (SEQ. ID (SEQ ID NO: 116) NO: 113)NO: 115) RL1 — — — QDIHGY YTS (SEQ QQGNTLPWT (SEQ ID ID (SEQ ID NO: 117)NO: 119) NO: 120) RL2 — — — QDIHGY YTS (SEQ QQGNTLPWT (SEQ ID ID (SEQ IDNO: 117) NO: 119) NQ: 120) RL3 — — — QSISSY AAS (SEQ QQGNTLPWT (SEQ IDID (SEQ ID NO: 118) NO: 177) NQ: 120) RL4 — — — QDIHGY YTS (SEQQQGNTLPWT (SEQ ID ID (SEQ ID NO: 117) NO: 119) NQ: 120) RL5 — — — QDIHGYYTS (SEQ QQGNTLPWT (SEQ ID ID (SEQ ID NO: 117) NO: 119) NQ: 120)

In still yet another aspect of the present disclosure, provided hereinis a conjugate comprising an antibody or antigen-binding fragmentthereof and one or more immunomodulating oligonucleotides (P), whereinthe antibody or antigen-binding fragment is linked to one or more Q-tagpeptides (Q) comprising the amino acid sequence RPQGF (SEQ ID NO:47),wherein each immunomodulating oligonucleotide is linked to a Q-tagpeptide via an amide bond with the glutamine residue of the Q-tagpeptide and a linker (L) as shown in formula (A),

wherein:

-   -   each Q independently comprises a Q-tag peptide sequence RPQGF        (SEQ ID NO:47);    -   each L is independently a bond or a linker moiety

wherein m is an integer ranging from about 0 to about 50, and wherein

indicates the point of attachment to P, and

indicates the point of attachment to the rest of the conjugate connectedto Q via an amide bond with the glutamine residue;

and each P is independently an immunomodulating oligonucleotide havingthe structure

wherein

and

indicate the points of attachment within the oligonucleotide, andwherein

indicates the point of attachment to L;

wherein Ab comprises a heavy chain variable (VHI) domain and a lightchain variable (VL) domain, wherein the VH domain comprises CDR-H1,CDR-H2, and CDR-H3 sequences from a VH domain sequence

(SEQ ID NO: 65) QVQLLESGGGVVQPGGSLRLSCAASGFAFSIYDMNWVRQAPGKGLEWVSAISSGGGTTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHSGYGTHWGVLFAYWGRGTLVTVSS;

wherein the VL domain comprises CDR-L1, CDR-L2, and CDR-L3 sequencesfrom a VL domain sequence:

(SEQ ID NO: 68) DIQMTQSPSSLSASVGDRVTITCRASQDIHGYLNWYQQKPGKAPKLLIYYTSILHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQGNTLPWTFGQ GTKLEIK.

In some embodiments of the foregoing, the VL domain further comprises anamino acid substitution at the N92 residue (numbering starting at theN-terminus of the VL domain sequence). In certain embodiments whereinthe VL domain comprises an amino acid substitution at N92, the VL domaincomprises an amino acid substitution N92A. In certain other embodimentswherein the VL domain comprises an amino acid substitution at N92, theVL domain comprises an amino acid substitution N92L. In still certainother embodiments wherein the VL domain comprises an amino acidsubstitution at N92, the VL domain comprises an amino acid substitutionN92S. In some embodiments of the foregoing, the VL domain comprises a VLdomain sequence shown in Table 6, e.g., SEQ ID Nos: 73-91.

In still other aspects, provided herein is a conjugate comprising aprotein, at least one Q tag peptide sequence comprising a glutamineresidue, and at least one immunomodulatory oligonucleotide, wherein theQ-tag peptide sequence is naturally occurring or synthetic, and whereinthe immunomodulatory oligonucleotide is linked to the Q-tag via an amidebond with the glutamine residue, wherein at least one Q-tag peptidesequence is selected from the group consisting of SEQ ID NOs: 39-55.

In some embodiments, the immunomodulatory oligonucleotide has a sequenceselected from the group consisting of the oligonucleotides of Table 10and Table 12.

In some embodiments, the preferred immunomodulatory agent in theanti-CD22 conjugate is a toll-like receptor agonist selected from TLR1agonist, TLR2 agonist, TLR3 agonist, TLR4 agonist, TLR5 agonist, TLR6agonist, TLR7 agonist, TLR8 agonist, and TLR10 agonist. In someembodiments, the immunomodulatory agent is a toll-like receptor agonistselected from TLR7 agonist, TLR8 agonist, TLR7/TLR8 agonist.

In some embodiments, the immunomodulatory agent in the anti-CD22conjugate is a STING pathway agonist or modulator of upstream enzymesthat modulate STING (e.g., inhibitors of ENPP1, a phosphodiesterase thatnegatively regulates the STING pathway). STING (stimulator of interferongenes, also known as TMEM173, MITA, ERIS, and MPYS) is a transmembraneprotein localized to the ER that undergoes a conformational change inresponse to direct binding of cyclic dinucleotides (CDNs), resulting ina downstream signaling cascade involving TBK1 activation, IRF-3phosphorylation, and production of IFN-P and other cytokines. The STINGpathway in tumor-resident host antigen presenting cells is involved inthe induction of a spontaneous CD8+ T cell response against tumorassociated antigens. Activation of this pathway and the subsequentproduction of IFN-β also contributes to the anti-tumor effect. In someembodiments, the STING pathway agonist is ADU-S100. Additional STINGagonists and their uses are described in, for example, US20180028553,US20170319680, US20170298139, US20060040887, US20080286296,US20120041057, US20140205653, WO2014179335, WO 2014179760,US20150056224, WO 2016096174, WO 2017011444, WO 2017027645, and WO2017027646.

In some embodiments, the immunomodulatory agent in the anti-CD22conjugate is a RIG-I pathway agonist. RIG-I (retinoic acid-induciblegene-I) is a member of pattern-recognition receptors that initiates ahost's innate immune system to defend against pathogenic microbes inearly phases of infection. There are three members of the (RIG-I)-likereceptors family: RIG-I, MDA5 (melanoma differentiation factor 5), andLGP2 (laboratory of genetics and physiology 2), which are expressed inmost cell and tissue types. RIG-I functions as a cytoplasmic sensor forthe recognition of a variety of RNA viruses and subsequent activation ofdownstream signaling to drive type I IFN production and antiviral geneexpressions. Activated RIG-I recruits its downstream adaptor moleculeMAVS (also known as IPS-1, CARDIF, and VISA) through CARD-CARD-mediatedinteractions. The oligomeric RIG-I CARD assembly and the polymericformation of MAVS, together serve as a signaling platform for proteincomplexes that mediate the bifurcation of signaling into two branches.One branch recruits tumor necrosis factor receptor-associated factors(TRAF)-2/6 and the receptor-interacting protein 1 to subsequentlyactivate the IKK complex, resulting in NF-κB activation. The otherbranch signals through TRAF3 and activates the TANK/IKKγ/IKKϵ/TBK1complex, leading to the phosphorylation and dimerization of interferonregulator factors (IRF)-3 and -7. Liu et al., Front Immunol. 2017,7:662. Activation of this pathway contributes to the anti-tumor effect.In some embodiments, the RIG-I pathway agonist is RGT100. RIG-I agonistsand their uses are described in, for example, US20170057978,US20170258897, U.S. Pat. Nos. 9,381,208, 9,738,680, 9,650,427,WO2017173427, and WO2017011622.

III. Proteins with Q-Tag

In one aspect, provided herein is a protein comprising at least one Qtag peptide sequence comprising a glutamine residue. In someembodiments, the Q tag peptide sequence is naturally occurring orsynthetic. In certain embodiments, the Q tag peptide sequence is aninternal reactive glutamine exposed by an amino acid substitution. Infurther embodiments, the Q tag is fused to the C-terminus of the heavychain of the protein. In still further embodiments, at least one of theat least one Q tag peptide sequences is elected from the groupconsisting of SEQ ID NOs: 39-55.

In some embodiments the protein is an antibody or an antigen-bindingfragment thereof. In certain embodiments, the antibody comprises a lightchain variable domain (VL) and a heavy chain variable domain (VH), andwherein VH comprises the sequence SEQ ID NO: 56; and VL comprises thesequence SEQ ID NO: 57.

In another aspect of the present disclosure, provided herein areantibodies of formula (B)

wherein:

each Q is independently a Q-tag comprising a peptide sequence with atleast one glutamine residue;

Ab is an antibody or antigen-binding fragment thereof, and

e is an integer from 1 to 20.

The antibodies of formula (B) may be precursors to theantibody-oligonucleotide conjugates of formula (A) as described above.Accordingly, the properties and embodiments of the antibodies asdescribed in the previous aspect of formula (A) may be the same ordifferent from the properties and/or embodiments of the antibodies offormula (B).

In some embodiments of the present aspect, the antibody or fragmentthereof is a monoclonal antibody or fragment thereof. In certainembodiments, the antibody or fragment thereof is a Fab, F(ab′)2,Fab′-SH, Fv, scFv, single domain, single heavy chain, or single lightchain antibody or antibody fragment. In other embodiments, the antibodyor fragment thereof is a humanized, human, or chimeric antibody orfragment thereof. In certain embodiments, which may be combined with anyof the preceding embodiments, the antibody or fragment thereofspecifically binds human CD22.

In some embodiments, wherein the antibody comprises a heavy chainvariable (VH) domain and a light chain variable (VL) domain, wherein theVH domain comprises CDR-H1, CDR-H2, and CDR-H3 sequences from a VHdomain sequence selected from the group consisting of:

(SEQ ID NO: 64) EVQLVESGGGLVQPGGSLRLSCAASGFAFSIYDMSWVRQAPGKGLEWVAYISSGGGTTYYPDTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHSGYGTHWGVLFAYWGRGTLVTVSS, (SEQ ID NO: 65)QVQLLESGGGVVQPGGSLRLSCAASGFAFSIYDMNWVRQAPGKGLEWVSAISSGGGTTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHSGYGTHWGVLFAYWGRGTLVTVSS, (SEQ ID NO: 66)EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYEMNWVRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHSGYGTHWGVLFAYWGRGTLVTVSS, and (SEQ ID NO: 67)QVQLQESGPGLVKPSDTLSLTCTVSGFAFSIYDMSWIRQPPGKGLEWIAYISSGGGTTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARHSGYGTHWGVLFAYWGRGTLVTVSS.

The antibody of any one of embodiments 87 to 90, wherein the antibodycomprises a heavy chain variable (VH) domain and a light chain variable(VL) domain, and wherein the VH domain comprises an amino acid sequenceselected from the group consisting of:

(SEQ ID NO: 64) EVQLVESGGGLVQPGGSLRLSCAASGFAFSIYDMSWVRQAPGKGLEWVAYISSGGGTTYYPDTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHSGYGTHWGVLFAYWGRGTLVTVSS, (SEQ ID NO: 65)QVQLLESGGGVVQPGGSLRLSCAASGFAFSIYDMNWVRQAPGKGLEWVSAISSGGGTTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHSGYGTHWGVLFAYWGRGTLVTVSS, (SEQ ID NO: 66)EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYEMNWVRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHSGYGTHWGVLFAYWGRGTLVTVSS, and (SEQ ID NO: 67)QVQLQESGPGLVKPSDTLSLTCTVSGFAFSIYDMSWIRQPPGKGLEWIAYISSGGGTTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARHSGYGTHWGVLFAYWGRGTLVTVSS.

The antibody of any one of embodiments 87 to 93, wherein the antibodycomprises a heavy chain variable (VH) domain and a light chain variable(VL) domain, wherein the VL domain comprises CDR-L1, CDR-L2, and CDR-L3sequences from a VL domain sequence selected from the group consistingof:

(SEQ ID NO: 68) DIQMTQSPSSLSASVGDRVTITCRASQDIHGYLNWYQQKPGKAPKLLIYYTSILHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQGNTLPWTFGQ GTKLEIK,(SEQ ID NO: 69) DIQMTQSPSSVSASVGDRVTITCRASQDIHGYLAWYQQKPGKAPKLLIYYTSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNTLPWTFGQ GTKLEIK,(SEQ ID NO: 70) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNTLPWTFGQ GTKLEIK,(SEQ ID NO: 71) EIVLTQSPATLSLSPGERATLSCRASQDIHGYLNWYQQKPGQAPRLLIYYTSILHSGIPARFSGSGPGTDFTLTISSLEPEDFAVYYCQQGNTLPWTFGG GTKLEIK, and(SEQ ID NO: 72) DIVMTQTPLSLSVTPGQPASISCRASQDIFIGYLNWYQQKPGQSPQLLIYYTSILHSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCQQGNTLPWTFG GGTKLEIK.

In some embodiments of the foregoing, the VL domain further comprises anamino acid substitution at residue N92. In certain embodiments of theforegoing, the VL domain comprises an amino acid substitution at residueN92 selected from the group consisting of N92A, N92L and N92S.

In some embodiments, the antibody comprises a heavy chain variable (VH)domain and a light chain variable (VL) domain, wherein the VL domain anamino acid sequence selected from the group consisting of:

(SEQ ID NO: 68) DIQMTQSPSSLSASVGDRVTITCRASQDIHGYLNWYQQKPGKAPKLLIYYTSILHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQGNTLPWTFGQ GTKLEIK,(SEQ ID NO: 69) DIQMTQSPSSVSASVGDRVTITCRASQDIHGYLAWYQQKPGKAPKLLIYYTSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNTLPWTFGQ GTKLEIK,(SEQ ID NO: 70) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNTLPWTFGQ GTKLEIK,(SEQ ID NO: 71) EIVLTQSPATLSLSPGERATLSCRASQDIHGYLNWYQQKPGQAPRLLIYYTSILHSGIPARFSGSGPGTDFTLTISSLEPEDFAVYYCQQGNTLPWTFGG GTKLEIK, and(SEQ ID NO: 72) DIVMTQTPLSLSVTPGQPASISCRASQDIFIGYLNWYQQKPGQSPQLLIYYTSILHSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCQQGNTLPWTFG GGTKLEIK.

In some embodiments, the antibody comprises an Fc region. In certainembodiments wherein the antibody comprises an Fc region, the Fc regionis a human Fc region selected from the group consisting of an IgG1 Fcregion, an IgG2 Fc region, and an IgG4 Fc region.

In certain embodiments of the present aspect, the Fc region is:

(a) a human IgG1 Fc region comprising L234A, L235A, and/or G237Asubstitutions, amino acid position numbering according to EU index;

(b) a human IgG2 Fc region comprising A330S and/or P331S substitutions,amino acid position numbering according to EU index; or

(c) a human IgG4 Fc region comprising S228P and/or L235E substitutions,amino acid position numbering according to EU index.

In some embodiments, the Fc region further comprises an N297Asubstitution, amino acid position numbering according to EU index. Inother embodiments, the Fc region further comprises a D265A substitution,amino acid position numbering according to EU index. In yet furtherembodiments, the antibody comprises a human lambda light chain. In otherembodiments, the antibody comprises a human kappa light chain.

In some embodiments, at least one Q-tag is attached to the heavy chainof the antibody. In certain embodiments, at least one Q-tag is fused tothe C-terminus of the heavy chain of the antibody. In other embodiments,at least one Q-tag is attached to the light chain of the antibody. Instill further embodiments, at least one Q-tag is within the Fe domain.

In some embodiments of the present aspect, the antibody is linked tofrom 1 to 20 Q-tags Q. In certain embodiments, the number of Q-tagslinked to the antibody/conjugate is an integer of about 1, about 2,about 3, about 4, about 5, about 6, about 7 about 8, about 9, about 10,about 11 about 12, about 13, about 14, about 15, about 16, about 17,about 18, about 19, or about 20. In certain other embodiments, 1 or 2Q-tags is/are linked to the antibody or antigen-binding fragment. In yetother embodiments, the number of Q-tags linked to the antibody/conjugateis an integer from 1 to 10, from 10 to 20, from 5 to 10, from 10 to 15,from 15 to 20, or from 1 to 5.

In still further embodiments of the present aspect, which may becombined with any of the preceding embodiments, each Q tag independentlycomprises or is a peptide sequence selected from the group consisting ofSEQ ID NOs: 39-55. In some embodiments, each Q tag independentlycomprises or is a peptide sequence selected from the group consisting ofthe peptide sequences of Table 3. In other embodiments of the presentaspect, each Q tag independently comprises or is a peptide sequenceselected from the group consisting of SEQ ID NOs: 40-55. In yet otherembodiments, each Q tag independently comprises or is a peptide sequenceselected from the group consisting of SEQ ID NOs: 47-49. In someembodiments, the Q-tag comprises LLQGG (SEQ ID NO:172), GGGLLQGG (SEQ IDNO: 173), RPQGF (SEQ ID NO:47), or RPQGFGPP (SEQ ID NO:49). In someembodiments of the present aspect, each Q is independently a Q-tagcomprising a peptide sequence RPQGF (SEQ ID NO:47). In certainembodiments, each Q-tag comprising a peptide sequence RPQGF (SEQ IDNO:47) is selected from the group consisting of RPQGF (SEQ ID NO:47),RPQGFPP (SEQ ID NO:48), and RPQGFGPP (SEQ ID NO:49). In certainembodiments, each Q tag independently comprises or is a peptide sequenceRPQGFGPP (SEQ ID NO:49).

IV. Immunomodulating Polynucleotides

In yet another aspect, provided herein is an immunomodulatingpolynucleotide of formula (C),

wherein

and

indicate the points of attachment within the oligonucleotide;

each T¹ is independently O or S;

each T² is S⁻;

T³ is a group

wherein

indicates the point of attachment to the rest of the oligonucleotide;

Z is O or S;

U^(5′) is —H or halogen;

R^(5′) is —H or methoxy;

R^(c1) is —H or methoxy;

R^(g1), R^(g2), R^(g3), and R^(g4) are H or oxo, provided that at leastone of R^(g1), R^(g2), R^(g3), and R^(g4) is oxo and wherein the carbonto which the oxo is attached has a single bond to the ring nitrogen atthe 7-position);

R^(3′) is methoxy or 2-methoxyethoxy;

R¹ is —(CH₂)₃—OH;

R² is —H or methyl; and

n is an integer from 0 to 2,

or a pharmaceutically acceptable salt thereof.

In some embodiments of the present aspect, U^(5′) is —H In otherembodiments, U^(5′) is halogen. In certain embodiments, U^(5′) is iodoor bromo. In some embodiments of the present aspect, theimmunomodulatory oligonucleotide of formula (C) is an immunomodulatoryoligonucleotide of formula (C′). In other embodiments of the presentaspect, the immunomodulatory oligonucleotide of formula (C) is animmunomodulatory oligonucleotide of formula (C″).

In some embodiments of the present aspect, provided herein is animmunomodulatory oligonucleotide of formula (C′)

wherein

and

indicate the points of attachment within the oligonucleotide;

each T¹ is independently O or S;

each T² is S⁻;

T³ is a group

wherein

indicates the point of attachment to the rest of the oligonucleotide;

Z is O or S;

R^(5′) is —H or methoxy;

R^(c1) is —H or methoxy;

R^(g1), R^(g2), R^(g3), and R^(g4) are H or oxo, provided that at leastone of R^(g1), R^(g2), R^(g3), and R^(g4) is oxo and wherein the carbonto which the oxo is attached has a single bond to the ring nitrogen atthe 7-position);

R^(3′) is methoxy or 2-methoxyethoxy;

R¹ is —(CH₂)₃—OH;

R² is —H or methyl; and

n is an integer from 0 to 2,

or a pharmaceutically acceptable salt thereof.

In other embodiments of the present aspect, provided herein is animmunomodulatory oligonucleotide of formula (C″)

wherein

and

indicate the points of attachment within the oligonucleotide;

each T¹ is independently O or S;

each T² is S⁻;

T³ is a group

wherein

indicates the point of attachment to the rest of the oligonucleotide;

Z is O or S;

R^(5′) is —H or methoxy;

R^(c1) is —H or methoxy;

R^(g1), R^(g2), R^(g3), and R^(g4) are H or oxo, provided that at leastone of R^(g1), R^(g2), R^(g3), and R^(g4) is oxo and wherein the carbonto which the oxo is attached has a single bond to the ring nitrogen atthe 7-position);

R^(3′) is methoxy or 2-methoxyethoxy;

R¹ is —(CH₂)₃—OH;

R² is —H or methyl; and

n is an integer from 0 to 2,

or a pharmaceutically acceptable salt thereof.

In some embodiments of the present aspect, provided herein is animmunomodulatory oligonucleotide of formula (C′)

wherein:

and

indicate the points of attachment within the oligonucleotide;

each T^(N) is independently O or S;

each T² is S⁻;

T³ is a group

wherein

indicates the point of attachment to the rest of the oligonucleotide;

Z is O or S;

R^(5′) is —H or methoxy;

R^(c1) is —H or methoxy;

R^(g1), R^(g2), R^(g3), and R^(g4) are H;

R^(3′) is methoxy;

R¹ is —(CH₂)₃—OH;

R² is —H or methyl; and

n is an integer from 0 to 2,

or a pharmaceutically acceptable salt thereof.

In other embodiments of the present aspect, provided herein is animmunomodulatory oligonucleotide of formula (C″)

wherein:

and

indicate the points of attachment within the oligonucleotide;

each T¹ is independently O or S;

each T² is S⁻;

T³ is a group

wherein

indicates the point of attachment to the rest of the oligonucleotide;

Z is O or S;

R^(5′) is —H or methoxy;

R^(c1) is —H or methoxy;

R^(g1), R^(g2), R^(g3), and R^(g4) are H;

R^(3′) is methoxy;

R¹ is —(CH₂)₃—OH;

R² is —H or methyl; and

n is an integer from 0 to 2,

or a pharmaceutically acceptable salt thereof.

In some embodiments of the present aspect, Z is S. In additionalembodiments, the oligonucleotide comprises at least one pair of geminalT¹ and T² wherein T¹ is S and T² is S⁻. In certain embodiments, theoligonucleotide comprises at least two pairs of geminal T¹ and T²wherein T¹ is S and T² is S. The pair(s) of geminal T¹ and T² wherein T¹is S and T² is S⁻ may also be described as phosphorodithioate linkages.

It should be recognized that in some instances wherein theoligonucleotide has at least one pair of geminal T¹ and T² wherein T¹ isS and T² is S⁻, the phosphorodithioate linkage(s) may be furtherdescribed in terms of the position within the oligonucleotide at whichthe linkage is located. The position of the linkage may becharacterized, for example, as being between two nucleoside residues,e.g., between the first and second nucleoside residues (or betweennucleoside residues 1 and 2) as counted from the 5′ end of theoligonucleotide. Alternatively, the position of the linkage may bedescribed as being located at the 3′-position of a given nucleosideresidue, e.g., on the internucleoside linker immediately following thespecified nucleoside residue or the 3′-position of the ′3-terminalresidue.

In some embodiments wherein the oligonucleotide comprises at least onepair of geminal T¹ and T² wherein T¹ is S and T² is S⁻, and wherein n is0, the at least one phosphorodithioate linkage is between nucleosideresidues 1 and 2, between nucleoside residues 2 and 3, betweennucleoside residues 3 and 4, between nucleoside residues 5 and 6,between nucleoside residues 6 and 7, between nucleoside residues 7 and8, between nucleoside residues 8 and 9, between nucleoside residues 9and 10, between nucleoside residues 10 and 11, or between nucleosideresidues 11 and 12. In some embodiments wherein the oligonucleotidecomprises at least one pair of geminal T¹ and T² wherein T¹ is S and T²is S⁻, and wherein n is 0, the at least one phosphorodithioate linkageis located at the 3′-position of nucleoside residue 1, nucleosideresidue 2, nucleoside residue 3, nucleoside residue 5, nucleosideresidue 6, nucleoside residue 7, nucleoside residue 8, nucleosideresidue 9, nucleoside residue 10, nucleoside residue 11, nucleosideresidue 12, or nucleoside residue 13.

In some embodiments wherein the oligonucleotide comprises at least onepair of geminal T¹ and T² wherein T¹ is S and T² is S⁻, and wherein n is1, the at least one phosphorodithioate linkage is between nucleosideresidues 1 and 2, between nucleoside residues 2 and 3, betweennucleoside residues 3 and 4, between nucleoside residues 5 and 6,between nucleoside residues 6 and 7, between nucleoside residues 7 and8, between nucleoside residues 8 and 9, between nucleoside residues 9and 10, between nucleoside residues 10 and 11, between nucleosideresidues 11 and 12, or between nucleoside residues 12 and 13. In someembodiments wherein the oligonucleotide comprises at least one pair ofgeminal T¹ and T² wherein T¹ is S and T² is S⁻, and wherein n is 0, theat least one phosphorodithioate linkage is located at the 3′-position ofnucleoside residue 1, nucleoside residue 2, nucleoside residue 3,nucleoside residue 5, nucleoside residue 6, nucleoside residue 7,nucleoside residue 8, nucleoside residue 9, nucleoside residue 10,nucleoside residue 11, nucleoside residue 12, nucleoside residue 13, ornucleoside residue 14.

In some embodiments wherein the oligonucleotide comprises at least onepair of geminal T¹ and T² wherein T¹ is S and T² is S⁻, and wherein n is1, the at least one phosphorodithioate linkage is between nucleosideresidues 1 and 2, between nucleoside residues 2 and 3, betweennucleoside residues 3 and 4, between nucleoside residues 5 and 6,between nucleoside residues 6 and 7, between nucleoside residues 7 and8, between nucleoside residues 8 and 9, between nucleoside residues 9and 10, between nucleoside residues 10 and 11, between nucleosideresidues 11 and 12, or between nucleoside residues 12 and 13. In someembodiments wherein the oligonucleotide comprises at least one pair ofgeminal T¹ and T² wherein T¹ is S and T² is S⁻, and wherein n is 1, theat least one phosphorodithioate linkage is located at the 3′-position ofnucleoside residue 1, nucleoside residue 2, nucleoside residue 3,nucleoside residue 5, nucleoside residue 6, nucleoside residue 7,nucleoside residue 8, nucleoside residue 9, nucleoside residue 10,nucleoside residue 11, nucleoside residue 12, nucleoside residue 13, ornucleoside residue 14.

In some embodiments wherein the oligonucleotide comprises at least onepair of geminal T¹ and T² wherein T¹ is S and T² is S⁻, and wherein n is2, the at least one phosphorodithioate linkage is between nucleosideresidues 1 and 2, between nucleoside residues 2 and 3, betweennucleoside residues 3 and 4, between nucleoside residues 5 and 6,between nucleoside residues 6 and 7, between nucleoside residues 7 and8, between nucleoside residues 8 and 9, between nucleoside residues 9and 10, between nucleoside residues 10 and 11, between nucleosideresidues 11 and 12, between nucleoside residues 12 and 13, or betweenresidues 13 and 14. In some embodiments wherein the oligonucleotidecomprises at least one pair of geminal T¹ and T² wherein T¹ is S and T²is S⁻, and wherein n is 2, the at least one phosphorodithioate linkageis located at the 3′-position of nucleoside residue 1, nucleosideresidue 2, nucleoside residue 3, nucleoside residue 5, nucleosideresidue 6, nucleoside residue 7, nucleoside residue 8, nucleosideresidue 9, nucleoside residue 10, nucleoside residue 11, nucleosideresidue 12, nucleoside residue 13, nucleoside residue 14, or residue 15.

In still other embodiments wherein the oligonucleotide has at least twophosphorodithioate linkages or comprises at least two pairs of geminalT¹ and T² wherein T¹ is S and T² is S⁻, the positions of one or bothphosphorodithioate linkages or pairs of T¹ and T² may be specified. Itshould be recognized that the positions of one or bothphosphorodithioate linkages may be independently varied.

In some embodiments of the present aspect, provided herein is animmunomodulatory oligonucleotide of formula (C′)

wherein:

and

indicate the points of attachment within the oligonucleotide;

each T¹ is independently O or S;

each T² is S⁻;

T³ is a group

wherein

indicates the point of attachment to the rest of the oligonucleotide;

Z is O or S;

R^(5′) is —H or methoxy;

R^(c1) is —H or methoxy;

R^(g1), R^(g2), R^(g3), and R^(g4) are H;

R^(3′) is methoxy;

R¹ is —(CH₂)₃—OH;

R² is —H or methyl; and

n is an integer from 0 to 2,

or a pharmaceutically acceptable salt thereof.

In other embodiments of the present aspect, provided herein is animmunomodulatory oligonucleotide of formula (C″)

wherein:

and

indicate the points of attachment within the oligonucleotide;

each T¹ is independently O or S;

each T² is S⁻;

T³ is a group

wherein

indicates the point of attachment to the rest of the oligonucleotide;

Z is O or S;

R^(5′) is —H or methoxy;

R^(c1) is —H or methoxy;

R^(g1), R^(g2), R^(g3), and R^(g4) are H;

R^(3′) is methoxy;

R¹ is —(CH₂)₃—OH;

R² is —H or methyl; and

n is an integer from 0 to 2,

or a pharmaceutically acceptable salt thereof.

In some embodiments of the present aspect, Z is S. In additionalembodiments, the oligonucleotide comprises at least one pair of geminalT¹ and T² wherein T¹ is S and T² is S⁻. In certain embodiments, theoligonucleotide comprises at least two pairs of geminal T¹ and T²wherein T¹ is S and T² is S⁻.

In still yet another embodiment of the present aspect, provided hereinis an oligonucleotide of formula (C)

wherein

and

indicate the points of attachment within the oligonucleotide;each T¹ is independently O or S;each T² is S⁻;provided that the oligonucleotide comprises at least one pair of geminalT¹ and T² wherein T¹ is S and T² is S,T³ is a group

wherein

indicates the point of attachment to the rest of the oligonucleotide;

Z is O or S;

U^(5′) is —H or halogen;

R^(5′) is —H; R^(c1) is —H;

R^(g1), R^(g2), R^(g3), and R^(g4) are H;R^(3′) is methoxy;R¹ is —(CH₂)₃—OH;R² is -methyl; andn is 1,or a pharmaceutically acceptable salt thereof.

In some embodiments of any of the foregoing, the at least one pair ofgeminal T¹ and T² wherein T¹ is S and T² is S is between nucleosideresidues 2 and 3, between nucleoside residues 3 and 4, betweennucleoside residues 5 and 6, between nucleoside residues 6 and 7,between nucleoside residues 7 and 8, between nucleoside residues 8 and9, between nucleoside residues 9 and 10, or between nucleoside residues10 and 11. In still other embodiments of the foregoing, theoligonucleotide comprises at least two pairs of of geminal T¹ and T²wherein T¹ is S and T² is S, and wherein the at least two pairs of ofgeminal T¹ and T² wherein T¹ is S and T² is S are between nucleosideresidues 2 and 3, between nucleoside residues 3 and 4, betweennucleoside residues 5 and 6, between nucleoside residues 6 and 7,between nucleoside residues 7 and 8, between nucleoside residues 8 and9, between nucleoside residues 9 and 10, or between nucleoside residues10 and 11.

In some embodiments, the oligonucleotide comprises one or two pairs ofgeminal T¹ and T² wherein T¹ is S and T² is S, and wherein the one ortwo pairs of geminal T¹ and T² are between nucleoside residues 2 and 3,between nucleoside residues 3 and 4, between nucleoside residues 5 and6, between nucleoside residues 6 and 7, between nucleoside residues 7and 8, between nucleoside residues 8 and 9, between nucleoside residues9 and 10, or between nucleoside residues 10 and 11. In certainembodiments, the oligonucleotide comprises one pair of geminal T¹ and T²wherein T¹ is S and T² is S, and wherein the pair of geminal T¹ and T²is between nucleoside residues 2 and 3, between nucleoside residues 3and 4, between nucleoside residues 5 and 6, between nucleoside residues6 and 7, between nucleoside residues 7 and 8, between nucleosideresidues 8 and 9, between nucleoside residues 9 and 10, or betweennucleoside residues 10 and 11. In certain other embodiments, theoligonucleotide comprises two pairs of geminal T¹ and T² wherein T¹ is Sand T² is S, and wherein the two pairs of geminal T¹ and T² wherein T¹is S and T² is S are between nucleoside residues 2 and 3, betweennucleoside residues 3 and 4, between nucleoside residues 5 and 6,between nucleoside residues 6 and 7, between nucleoside residues 7 and8, between nucleoside residues 8 and 9, between nucleoside residues 9and 10, or between nucleoside residues 10 and 11.

In some embodiments, R^(5′) is H. In other embodiments, R^(5′) ismethoxy. In some embodiments, R^(c1) is H. In yet other embodiments,R^(c1) is methoxy. In still further embodiments, R² is methyl. In stillother embodiments, R² is H. In yet other additional embodiments, whichmay be combined with any of the preceding embodiments, T³ is

In still other embodiments, T³ is

In certain embodiments, m is an integer from 20 to 25.

In another aspect, the immunomodulating oligonucleotide of formula (C)is an oligonucleotide selected from the group consisting of theoligonucleotides of Table 9 and Table 10, or a pharmaceuticallyacceptable salt thereof. In still other embodiments, theimmunomodulating oligonucleotide of formula (C) is an oligonucleotideselected from the group consisting of the oligonucleotides of Table 10,or a pharmaceutically acceptable salt thereof.

TABLE 9 Modified Oligonucleotide Structures (with PEG₃NH₂) Cmpd #Structure 1.1a

2.1a

2.2a

2.3a

2.4a

TABLE 10 Modified Oligonucleotide Structures (with -PEG₃NH₂) Cmpd #Structure 3.1a

3.2a

3.3a

4.1a

4.2a

4.3a

5.1a

5.2a

5.3a

5.4a

5.5a

5.6a

5.7a

5.8a

5.9a

5.10a

5.11a

5.12a

6.1a

6.2a

6.3a

7.1a

7.2a

7.3a

7.4a

7.5a

7.6a

7.7a

7.8a

7.9a

7.10a

15.7a

In some embodiments, the immunomodulating oligonucleotides of formula(C) may be utilized without conjugation to an antibody orantigen-binding fragment thereof or may be used as precursors to prepareconjugates comprising an antibody or antigen-binding fragment thereofand one or more immunomodulating oligonucleotides of formula (C) linkedvia Q-tag as shown in the structures of formula (A) as described herein.

In one aspect, provided herein is an immunomodulating oligonucleotide offormula (C), wherein the oligonucleotide is not conjugated to anydelivery modality (such as a nanoparticle or protein) or targetingmoiety (such as an antibody or antigen-fragment thereof). Sucholigonucleotides may be further referred to as “naked” oligonucleotidesor “naked” CpGs.

In another aspect, provided herein are immunomodulating oligonucleotidesof formula (C), wherein the immunomodulating oligonucleotide ispegylated. In a further aspect, provided herein are immunomodulatingoligonucleotides of formula (C), wherein the immunomodulatingoligonucleotide is immobilized on a bead. In yet another aspect,provided herein are immunomodulating oligonucleotides of formula (C),wherein the immunomodulating oligonucleotide is formulated in ananoparticle. In still a further aspect, provided herein areimmunomodulating oligonucleotides of formula (C), wherein theimmunomodulating oligonucleotide is encapsulated in a liposome. In yet afurther aspect, provided herein are immunomodulating oligonucleotides offormula (C), wherein the immunomodulating oligonucleotide is conjugatedto a polypeptide.

In still other aspect, provided herein is a method for delivering theimmunomodulating oligonucleotide according to any of the embodimentsherein, comprising contacting the immunomodulating oligonucleotide witha cell. In some embodiments, the immunomodulating oligonucleotide ispegylated. In other embodiments, the immunomodulating oligonucleotide isimmobilized on a bead. In some embodiments, the immunomodulatingoligonucleotide is formulated in a nanoparticle. In still otherembodiments, the immunomodulating oligonucleotide is encapsulated in aliposome. In some embodiments, the immunomodulating oligonucleotide isconjugated to a polypeptide.

In still further embodiments, the immunomodulating oligonucleotides offormula (C) may be modified to attach a linker moiety L to the terminalgroup T³ in formula (C) to provide immunomodulating oligonucleotides offormula (D). In still another aspect, provided herein areimmunomodulating oligonucleotides of formula (D)

wherein

and

indicate the points of attachment within the oligonucleotide;

each T¹ is independently O or S;

each T² is S⁻;

T³ is a group

wherein

indicates the point of attachment to L and wherein

indicates the point of attachment to the rest of the oligonucleotide;

L is a group

wherein m is an integer from 0 to 50 and wherein

indicates the point of attachment to the rest of the oligonucleotide viaT³;

Z is O or S;

U^(5′) is —H or halogen;

R^(5′) is —H or methoxy;

R^(c1) is —H or methoxy;

R^(g1), R^(g2), R^(g3), and R^(g4) are H or oxo, provided that at leastone of R^(g1), R^(g2), R^(g3), and R^(g4) is oxo and wherein the carbonto which the oxo is attached has a single bond to the ring nitrogen atthe 7-position;

R^(3′) is methoxy or 2-methoxyethoxy;

R¹ is —(CH₂)₃—OH;

R² is —H or methyl; and

n is an integer from 0 to 2,

or a pharmaceutically acceptable salt thereof.

In some embodiments of the present aspect, U^(5′) is —H In otherembodiments, U^(5′) is halogen. In certain embodiments, U^(5′) is iodoor bromo. In some embodiments of the present aspect, theimmunomodulatory oligonucleotide of formula (D) is an immunomodulatoryoligonucleotide of formula (D′). In other embodiments of the presentaspect, the immunomodulatory oligonucleotide of formula (D) is animmunomodulatory oligonucleotide of formula (D″).

In some embodiments of the present aspect, provided herein is animmunomodulatory oligonucleotide of formula (D′)

wherein

and

indicate the points of attachment within the oligonucleotide;

each T¹ is independently O or S;

each T² is S⁻;

T³ is a group

wherein

indicates the point of attachment to L and wherein

indicates the point of attachment to the rest of the oligonucleotide;

L is a group

wherein m is an integer from 0 to 50 and wherein

indicates the point of attachment to the rest of the oligonucleotide viaT³;

Z is O or S;

R^(5′) is —H or methoxy;

R^(c1) is —H or methoxy;

R^(g1), R^(g2), R^(g3), and R^(g4) are H or oxo, provided that at leastone of R^(g1), R^(g2), R^(g3), and R^(g4) is oxo and wherein the carbonto which the oxo is attached has a single bond to the ring nitrogen atthe 7-position;

R^(3′) is methoxy or 2-methoxyethoxy;

R¹ is —(CH₂)₃—OH;

R² is —H or methyl; and

n is an integer from 0 to 2,

or a pharmaceutically acceptable salt thereof.

In other embodiments of the present aspect, provided herein is animmunomodulatory oligonucleotide of formula (D″)

wherein

and

indicate the points of attachment within the oligonucleotide;

each T¹ is independently O or S;

each T² is S⁻;

T³ is a group

wherein

indicates the point of attachment to L and wherein

indicates the point of attachment to the rest of the oligonucleotide;

L is a group

wherein m is an integer from 0 to 50 and wherein

indicates the point of attachment to the rest of the oligonucleotide viaT³;

Z is O or S;

R^(5′) is —H or methoxy;

R^(c1) is —H or methoxy;

R^(g1), R^(g2), R^(g3), and R^(g4) are H or oxo, provided that at leastone of R^(g1), R^(g2), R^(g3), and R^(g4) is oxo and wherein the carbonto which the oxo is attached has a single bond to the ring nitrogen atthe 7-position;

R^(3′) is methoxy or 2-methoxyethoxy;

R¹ is —(CH₂)₃—OH;

R² is —H or methyl; and

n is an integer from 0 to 2,

or a pharmaceutically acceptable salt thereof.

In some embodiments of the present aspect, the present disclosure alsoprovides immunomodulating oligonucleotides of formula (D′)

wherein

and

indicate the points of attachment within the oligonucleotide;

each T¹ is independently O or S;

each T² is S⁻;

T³ is a group

wherein

indicates the point of attachment to L and wherein

indicates the point of attachment to the rest of the oligonucleotide;

L is a group

wherein m is an integer from 0 to 50 and wherein

indicates the point of attachment to the rest of the oligonucleotide viaT³;

Z is O or S;

R^(5′) is —H or methoxy;

R^(c1) is —H or methoxy;

R^(g1), R^(g2), R^(g3), and R^(g4) are H;

R^(3′) is methoxy;

R¹ is —(CH₂)₃—OH;

R² is —H or methyl; and

n is an integer from 0 to 2,

or a pharmaceutically acceptable salt thereof.

In still other embodiments of the present aspect, provided herein is animmunomodulatory oligonucleotide of formula (D″)

wherein

and

indicate the points of attachment within the oligonucleotide;

each T¹ is independently O or S;

each T² is S

T³ is a group

wherein

indicates the point of attachment to L and wherein

indicates the point of attachment to the rest of the oligonucleotide;

L is a group

wherein m is an integer from 0 to 50 and wherein

indicates the point of attachment to the rest of the oligonucleotide viaT³;

Z is O or S;

R^(5′) is —H or methoxy;

R^(c1) is —H or methoxy;

R^(g1), R^(g2), R^(g3), and R^(g4) are H;

R^(3′) is methoxy;

R¹ is —(CH₂)₃—OH;

R² is —H or methyl; and

n is an integer from 0 to 2,

or a pharmaceutically acceptable salt thereof.

In some embodiments of the present aspect, Z is S. In additionalembodiments, the oligonucleotide comprises at least one pair of geminalT¹ and T² wherein T¹ is S and T² is S. In certain embodiments, theoligonucleotide comprises at least two pairs of geminal T¹ and T²wherein T¹ is S and T² is S.

In still yet another embodiment of the present aspect, provided hereinis an oligonucleotide of formula (D)

wherein

and

indicate the points of attachment within the oligonucleotide;each T¹ is independently O or S;each T² is S⁻;provided that the oligonucleotide comprises at least one pair of geminalT¹ and T² wherein T¹ is S and T² is S,T³ is a group

wherein

indicates the point of attachment to L and wherein

indicates the point of attachment to the rest of the oligonucleotide;L is a group

wherein m is an integer from 0 to 50 and wherein

indicates the point of attachment to the rest of the oligonucleotide viaT³;

Z is O or S;

U^(5′) is —H or halogen;

R^(5′) is —H; R^(c1) is —H;

R^(g1), R^(g2), R^(g3), and R^(g4) are H;R^(3′) is methoxy;R¹ is —(CH₂)₃—OH;R² is -methyl; andn is 1,or a pharmaceutically acceptable salt thereof.

In some embodiments of any of the foregoing, the at least one pair ofgeminal T¹ and T² wherein T¹ is S and T² is S is between nucleosideresidues 2 and 3, between nucleoside residues 3 and 4, betweennucleoside residues 5 and 6, between nucleoside residues 6 and 7,between nucleoside residues 7 and 8, between nucleoside residues 8 and9, between nucleoside residues 9 and 10, or between nucleoside residues10 and 11. In still other embodiments of the foregoing, theoligonucleotide comprises at least two pairs of of geminal T¹ and T²wherein T¹ is S and T² is S, and wherein the at least two pairs of ofgeminal T¹ and T² wherein T¹ is S and T² is S are between nucleosideresidues 2 and 3, between nucleoside residues 3 and 4, betweennucleoside residues 5 and 6, between nucleoside residues 6 and 7,between nucleoside residues 7 and 8, between nucleoside residues 8 and9, between nucleoside residues 9 and 10, or between nucleoside residues10 and 11.

In some embodiments, the oligonucleotide comprises one or two pairs ofgeminal T¹ and T² wherein T¹ is S and T² is S, and wherein the one ortwo pairs of geminal T¹ and T² are between nucleoside residues 2 and 3,between nucleoside residues 3 and 4, between nucleoside residues 5 and6, between nucleoside residues 6 and 7, between nucleoside residues 7and 8, between nucleoside residues 8 and 9, between nucleoside residues9 and 10, or between nucleoside residues 10 and 11. In certainembodiments, the oligonucleotide comprises one pair of geminal T¹ and T²wherein T¹ is S and T² is S, and wherein the pair of geminal T¹ and T²is between nucleoside residues 2 and 3, between nucleoside residues 3and 4, between nucleoside residues 5 and 6, between nucleoside residues6 and 7, between nucleoside residues 7 and 8, between nucleosideresidues 8 and 9, between nucleoside residues 9 and 10, or betweennucleoside residues 10 and 11. In certain other embodiments, theoligonucleotide comprises two pairs of geminal T¹ and T² wherein T¹ is Sand T² is S, and wherein the two pairs of geminal T¹ and T² wherein T¹is S and T² is S are between nucleoside residues 2 and 3, betweennucleoside residues 3 and 4, between nucleoside residues 5 and 6,between nucleoside residues 6 and 7, between nucleoside residues 7 and8, between nucleoside residues 8 and 9, between nucleoside residues 9and 10, or between nucleoside residues 10 and 11.

In some embodiments, R^(5′) is H. In other embodiments, R^(5′) ismethoxy. In some embodiments, R^(c1) is H. In yet other embodiments,R^(c1) is methoxy. In still further embodiments, R² is methyl. In stillother embodiments, R² is H. In yet other additional embodiments, whichmay be combined with any of the preceding embodiments, T³ is

In still other embodiments, T³ is

In certain embodiments, m is an integer from 20 to 25.

In another aspect, the immunomodulating oligonucleotide of formula (D)is an oligonucleotide selected from the group consisting of theoligonucleotides of Table 11 and Table 12, or a pharmaceuticallyacceptable salt thereof. In still further embodiments of the presentaspect, the oligonucleotide of formula (D) is selected from the groupconsisting of the oligonucleotides of Table 12, or a pharmaceuticallyacceptable salt thereof.

TABLE 11 Modified Oligonucleotide Structures (with —PEG₃NHCOPEG₂₄NH₂)Cmpd # Structure 1.1b

2.1b

2.2b

2.3b

2.4b

TABLE 12 Modified Oligonucleotide Structures (with —PEG₃NHCOPEG₂₄NH₂)Cmpd # Structure  3.1b

 3.2b

 3.3b

 4.1b

 4.2b

 4.3b

 5.1b

 5.2b

 5.3b

 5.4b

 5.5b

 5.6b

 5.7b

 5.8b

 5.9b

 5.10b

 5.11b

 5.12b

 6.1b

 6.2b

 6.3b

 7.1b

 7.2b

 7.3b

 7.4b

 7.5b

 7.6b

 7.7b

 7.8b

 7.9b

 7.10b

15.7b

As with the oligonucleotides of formula (C), the immunomodulatingoligonucleotides of formula (D) may be utilized without conjugation toan antibody or antigen-binding fragment thereof or may be used asprecursors to prepare conjugates comprising an antibody orantigen-binding fragment thereof and one or more immunomodulatingoligonucleotides of formula (D) linked via Q-tag as shown in thestructures of formula (A) as described herein.

In one aspect, provided herein is an immunomodulating oligonucleotide offormula (D), wherein the oligonucleotide is not conjugated to anydelivery modality (such as a nanoparticle or protein) or targetingmoiety (such as an antibody or antigen-fragment thereof). Sucholigonucleotides may be further referred to as “naked” oligonucleotidesor “naked” CpGs.

In another aspect, provided herein are immunomodulating oligonucleotidesof formula (D), wherein the immunomodulating oligonucleotide ispegylated. In a further aspect, provided herein are immunomodulatingoligonucleotides of formula (D), wherein the immunomodulatingoligonucleotide is immobilized on a bead. In yet another aspect,provided herein are immunomodulating oligonucleotides of formula (D),wherein the immunomodulating oligonucleotide is formulated in ananoparticle. In still a further aspect, provided herein areimmunomodulating oligonucleotides of formula (D), wherein theimmunomodulating oligonucleotide is encapsulated in a liposome. In yet afurther aspect, provided herein are immunomodulating oligonucleotides offormula (D), wherein the immunomodulating oligonucleotide is conjugatedto a polypeptide.

In still other aspect, provided herein is a method for delivering theimmunomodulating oligonucleotide according to any of the embodimentsherein, comprising contacting the immunomodulating oligonucleotide witha cell. In some embodiments, the immunomodulating oligonucleotide ispegylated. In other embodiments, the immunomodulating oligonucleotide isimmobilized on a bead. In some embodiments, the immunomodulatingoligonucleotide is formulated in a nanoparticle. In still otherembodiments, the immunomodulating oligonucleotide is encapsulated in aliposome. In some embodiments, the immunomodulating oligonucleotide isconjugated to a polypeptide.

The immunomodulating oligonucleotides of formulae (C) and (D) asdescribed herein may be prepared according to methods known in the art.A general method for the preparation of immunomodulatingoligonucleotides, including those provided in the present disclosure, isdescribed below.

General Polynucleotide Synthesis:

Experimental Details

Automated polynucleotide synthesis (1 μmol scale) was carried out onMerMade 6 or 12 with the following reagents and solvents:

-   -   Oxidizer—0.02M I₂ in THF/pyridine/H₂O (60 s oxidation per        cycle),    -   Sulfurizing Reagent II—dithiazole        derivative/pyridine/acetonitrile (0.05 M, in 6:4        pyridine:acetonitrile) (60 s per cycle)    -   Deblock—3% trichloroacetic acid (2×40 s deblocks per cycle),    -   Cap Mix A—THF/2,6-lutidine/Ac20 (60 s capping per cycle), and    -   Cap Mix B—16% methyl imidazole in THE (60 s capping per cycle)

Exceptions to standard polynucleotide synthesis conditions were asfollows:

-   -   CPG supports with a non-nucleosidic linker called Uny-linker was        used.    -   All 2′-deoxyribose-phosphoramidites were resuspended to 100 mM        in 100% anhydrous acetonitrile prior to synthesis, except some        of the modified 2′-deoxy-phosphoramidites were dissolved to 100        mM in THF/acetonitrile mixture (1:4) depend on the solubility of        the starting material.    -   Phosphoramidite activation was performed with a 2.5-fold molar        excess of 5-benzylthio-1H-tetrazole (BTT). Activated        2′-deoxyribose-phosphoramidites were coupled for 2×1 minute        coupling per insertion and modified phosphoramidites were        coupled for 2×3 minute coupling per insertion.    -   Sulfurization of the backbone was performed with 0.05M        Sulfurizing Reagent II in pyridine/acetonitrile (6:4) for 1 min.

Polynucleotide Deprotection & Purification Protocol:

Following automated polynucleotide synthesis, solid support and baseprotecting groups (such as A-Bz, C-Ac, G-iBu, etc.) and methyl esters ofphosphotriesters were cleaved and de-protected in 1 mL of AMA (1:1 ratioof 36% aq. ammonia and 40% methylamine in methanol) for 2 h or more atroom temperature followed by centrifugal evaporation.

Crude polynucleotide pellets were resuspended in 100 μL of 50%acetonitrile, briefly heated to 65° C. and vortexed thoroughly.

For polynucleotide purification, 100 μL crude polynucleotides wereinjected onto RP-HPLC with the following buffers/gradient:

-   -   Buffer A=50 mM TEAA in Water;    -   Buffer B=90% Acetontrile; and    -   Flow Rate=1 mL/min;    -   Gradient:        -   0-2 min (100% Buffer A/0% Buffer B),        -   2-42 min (0% to 60% Buffer B), and        -   42-55 min (60% to 100% Buffer B).

DBCO Conjugation and Purification Protocol:

DBCO NHS ester was conjugated to the crude 2′-deoxy DMT-polynucleotideas described here. The crude polynucleotide pellet was suspended into 45μL DMSO, briefly heated to 65° C. and vortexed thoroughly. 5 μL of DIPEAwas added followed by DBCO-NHS ester (30 eq), which was pre-dissolved inDMSO (1M). The reaction was allowed to stand for 10 minutes or untilproduct formation was confirmed by MALDI. Total 80 μL of crudepolynucleotide samples were injected onto RP-HPLC with the followingbuffers/gradient:

-   -   Buffer A=50 mM TEAA in Water    -   Buffer B=90% Acetonitrile    -   Flow Rate=1 mL/min    -   Gradient:        -   0-2 min (90% Buffer A/10% Buffer B)        -   2-42 min (0% to 60% Buffer B)        -   42-55 min (60% to 100% Buffer B).

Across the dominant RP-HPLC peaks, 0.5 mL fractions were collected andanalyzed by MALDI-TOF mass spectrometry to confirm presence of desiredmass. Mass-selected, purified fractions were frozen and lyophilized.Once dry, fractions were re-suspended, combined with correspondingfractions, frozen and lyophilized.

DMT Cleavage: lyophilized pellets were suspended in 20 μL of 50%acetonitrile and added 80 μL of acetic acid, samples were kept standingat room temperature for 1 h, frozen and lyophilized. The dried sampleswere re-dissolved in 20% acetonitrile and desalted through NAP 10(Sephadex™-G25 DNA Grade) columns. Collected, pure fractions were frozenand lyophilized for final product.

Methods for Attaching Oligonucleotides to Linking Moiety Cu-CatalyzedClick Reaction

Copper-THPTA Complex Preparation

A 5 mM aqueous solution of copper sulfate pentahydrate (CuSO₄-5H₂O) anda 10 mM aqueous solution of tris(3-hydroxypropyltriazolylmethyl)amine(THPTA) were mixed 1:1 (v/v) (1:2 molar ratio) and allowed to stand atroom temperature for 1 hour. This complex can be used to catalyzeHuisgen cycloaddition, e.g., as shown in the general conjugation schemesbelow.

General Procedure (100 nM Scale):

To a solution of 710 μL of water and 100 μL tert-butanol (10% of finalvolume) in a 1.7 mL Eppendorf tube was added 60 μL of the copper-THPTAcomplex followed by 50 μL of a 2 mM solution of the oligo, 60 μL of a 20mM aqueous sodium ascorbate solution and 20 μL of a 10 mM solution oftargeting moiety-azide. After thorough mixing the solution was allowedto stand at room temperature for 1 hour. Completion of the reaction wasconfirmed by gel analysis. The reaction mixture is added to a screw capvial containing 5-10 fold molar excess of SiliaMetS® TAAcONa (resinbound EDTA sodium salt). The mixture is stirred for 1 hour. This mixtureis then eluted through an Illustra™Nap™-10 column Sephadex™. Theresulting solution is then frozen and lyophilized overnight.

Attachment Through Amide Linkage:

Conjugation through amidation may be performed under the amidationreaction conditions known in the art. See, e.g., Aaronson et al.,Bioconjugate Chem. 22:1723-1728, 2011.

where

each q is 0 or 1;

each m is an integer from 0 to 5;

Z is O or S;

R^(O) is a bond to a nucleoside in a polynucleotide;

R is a bond to H, a nucleoside in a polynucleotide, to solid support, orto a capping group (e.g., —(CH₂)₃—OH);

each R′ is independently H, -Q¹-Q^(A1) a bioreversible group, or anon-bioreversible group;

each R″ is independently H, -Qi-Q^(A)-Q¹-T, a bioreversible group, or anon-bioreversible group;

each R^(A) is independently H or —OR^(C), where R^(C) is -Q¹-Q^(A1) abioreversible group, or a non-bioreversible group;

each R^(B) is independently H or —OR^(D), where R^(D) is -Q¹-Q^(A)-Q²-T,a bioreversible group, or a non-bioreversible group;

where

-   -   each Q¹ is independently a divalent, trivalent, tetravalent, or        pentavalent group, in which one valency is bonded to Q^(A) or        Q^(A1), the second valency is open, and each of the remaining        valencies, when present, is independently bonded to an auxiliary        moiety;    -   each Q² is independently a divalent, trivalent, tetravalent, or        pentavalent group, in which one valency is bonded to Q^(A), the        second valency is bonded to T, and each of the remaining        valencies, when present, is independently bonded to an auxiliary        moiety;    -   Q^(A) is optionally substituted C₂₋₁₂ heteroalkylene containing        —C(O)—N(H)— or —N(H)—C(O)—;    -   Q^(A1) is —NHR^(N1) or —COOR¹², where R^(N1) is H, N-protecting        group, or optionally substituted C₁₋₆ alkyl, and R¹² is H,        optionally substituted C₁₋₆ alkyl, or O-protecting group; and    -   T is a linking moiety,    -   provided that the starting materials contain at least one        -Q¹-Q^(A1) and products contain -Q¹-Q^(A)-Q²-T.

Solution Phase Attachment:

where

m is an integer from 0 to 5;

Z is O or S;

R⁰ is a bond to a nucleoside in a polynucleotide;

R is a bond to H, a nucleoside in a polynucleotide, or to a cappinggroup;

each R′ is independently H, -Q¹-NH₂, a bioreversible group, or anon-bioreversible group;

each R″ is independently H, -Q¹-NH—CO-Q²-T, a bioreversible group, or anon-bioreversible group;

each R^(A) is independently H or —OR^(C), where R^(C) is -Q¹-NH₂, abioreversible group, or a non-bioreversible group;

each R^(B) is independently H or —OR^(D), where R^(D) is -Q¹-NH—CO-Q²-T,a bioreversible group, or a non-bioreversible group;

where

-   -   each Q¹ is independently a divalent, trivalent, tetravalent, or        pentavalent group, in which one valency is bonded to —NH—CO— or        —NH₂, the second valency is open, and each of the remaining        valencies, when present, is independently bonded to an auxiliary        moiety;    -   each Q² is independently a divalent, trivalent, tetravalent, or        pentavalent group, in which one valency is bonded to —NH—CO—,        the second valency is a bond to T, and each of the remaining        valencies, when present, is independently bonded to an auxiliary        moiety; and    -   T is a linking moiety,    -   provided that the starting material contains -Q¹-NH₂, and the        product contains -Q¹-NH—CO-Q²-T.

On-Support Attachment:

where

Z is O or S;

R^(O) is a bond to a nucleoside in a polynucleotide; each Q² isindependently a divalent, trivalent, tetravalent, or pentavalent group,in which one valency is bonded to —NH—CO—, the second valency is a bondto T, and each of the remaining valencies, when present, isindependently bonded to an auxiliary moiety; and

T is a linking moiety.

where

n is an integer from 1 to 8;

A is O or —CH₂—;

Z is O or S;

R^(O) is a bond to a nucleoside in a polynucleotide;

each Q² is independently a divalent, trivalent, tetravalent, orpentavalent group; in which one valency is bonded to the azide ortriazole, a second valency is bonded to T, and each of the remainingvalencies, when present, is independently bonded to an auxiliary moiety;and

T is a linking moiety.

where

n is an integer from 1 to 8;

A is O or —CH₂—;

Z is O or S;

R^(O) is a bond to a nucleoside in a polynucleotide;

each Q² is independently a divalent, trivalent, tetravalent, orpentavalent group; in which one valency is bonded to the azide ortriazole, a second valency is bonded to T, and each of the remainingvalencies, when present, is independently bonded to an auxiliary moiety;and

T is a linking moiety.

where

n is an integer from 1 to 8;

A is O or —CH₂—;

Z is O or S;

R^(O) is a bond to a nucleoside in a polynucleotide;

each Q² is independently a divalent, trivalent, tetravalent, orpentavalent group; in which one valency is bonded to the azide ortriazole, a second valency is bonded to T, and each of the remainingvalencies, when present, is independently bonded to an auxiliary moiety;and

each T is independently a linking moiety.

Representative Example of Fmoc Deprotection of a Phosphotriester:

A polynucleotide including a phosphotriester with Fmoc-protected aminewas subjected to deprotection conditions resulting in Fmoc deprotectionwithout observable conversion of the phosphotriester into aphosphodiester.

DBCO-NHS Conjugation to TCCATGACGTTCCTGACGTT (SEQ IDNO:176)-Representative Example:

DBCO-NHS conjugation to the amino group in the phosphotriester wascomplete in 10 min at room temperature, as evidenced by massspectrometric analysis.

RP-HPLC purification of TCCATGACGTTCCTGACGTT (SEQ ID NO:176) containinga DBCO conjugating group was performed using the following conditions:

-   -   Buffer A=50 mM TEAA in Water;    -   Buffer B=90% Acetontrile; and    -   Flow Rate=1 mL/min;    -   Gradient:        -   0-2 min (100% Buffer A/0% Buffer B),        -   2-22 min (0% to 100% Buffer B), and        -   22-25 min (100% Buffer B).

A similar procedure may be used to prepare a polynucleotide using, e.g.,2′-modified nucleoside phosphoramidites, such as those described herein.Such a procedure is provided in International Patent applicationPCT/US2015/034749; the disclosure of the disulfide phosphotriesteroligonucleotide synthesis in PCT/US2015/034749 is hereby incorporated byreference.

V. Methods of Conjugation

Provided herein are methods for preparing a conjugate comprising anantibody or antigen-binding fragment thereof and one or moreimmunomodulating oligonucleotides linked via one or more Q-tag peptidesas shown in the structure of Formula (A). In some embodiments, themethods comprise combining an antibody comprising at least one Q-tagpeptide sequence with at least one exposed glutamine residue and anoligonucleotide under conditions sufficient to induce conjugation, i.e.,amidation reaction between the CpG and Q tag. In other embodiments, themethods comprise reacting an antibody comprising at least one Q-tagpeptide sequence with at least one exposed glutamine residue and anoligonucleotide under chemical conditions sufficient to induceconjugation. In still other embodiments, the methods comprise reactingan antibody comprising at least one Q-tag peptide sequence with at leastone exposed glutamine residue and an oligonucleotide under enzymaticconditions, e.g., with transglutaminase, sufficient to induceconjugation.

Transglutaminase-Mediated Conjugation Reaction Conditions

In one aspect, provided herein is a method of preparing a conjugate offormula (A), comprising combining one or more immunomodulatingoligonucleotides (P) and an antibody comprising one or more glutamineresidues. In one aspect, provided herein is a method of preparing aconjugate comprising an antibody or antigen-binding fragment (Ab) andone or more immunomodulating oligonucleotides (P), wherein the antibodyor antigen-binding fragment is linked to one or more Q-tag peptides (Q)comprising the amino acid sequence RPQGF (SEQ ID NO:47), and whereineach immunomodulating oligonucleotide is linked to a Q-tag peptide viaan amide bond with the glutamine residue of the Q-tag peptide and alinker (L) as shown in formula (A),

wherein:

indicates the point of attachment of each Q to the antibody orantigen-binding fragment thereof (Ab);

each Q independently comprises a Q-tag peptide sequence RPQGF (SEQ IDNO:47);

each L is independently a bond or a linker moiety connected to Q via anamide bond with the glutamine residue; and

each P is independently an immunomodulating oligonucleotide; comprisingcontacting a compound of formula (B)

wherein Ab and Q are as defined for formula (A) above, and e is aninteger from 1 to 20, with one or more immunomodulating oligonucleotidesP, wherein each P independently has the following formula:

wherein

X^(5′) is a 5′ terminal nucleoside;

X^(3′) is a 3′ terminal nucleoside;

Y^(PTE) is an internucleoside phosphotriester;

Y^(3′) is a terminal phosphotriester;

each X^(N) is independently a nucleoside;

each Y^(N) is independently an internucleoside linker;

b and c are each independently an integer from 1 to 25; with the provisothat the sum of b and c is at least 5; and

L is a linker moiety having a terminal amine,

in the presence of a transglutaminase.

In another aspect, method for preparing a conjugate comprising anantibody or antigen-binding fragment (Ab) and one or moreimmunomodulating oligonucleotides (P), wherein the antibody orantigen-binding fragment is linked to one or more Q-tag peptides (Q)comprising at least one glutamine residue, and wherein eachimmunomodulating oligonucleotide is linked to a Q-tag peptide via anamide bond with the glutamine residue of the Q-tag peptide and a linker(L) as shown in Formula (A),

wherein:

indicates the point of attachment of each Q to the antibody orantigen-binding fragment thereof (Ab);

each Q is independently a Q-tag peptide having at least one glutamineresidue;

each L is independently a bond or a linker moiety connected to Q via anamide bond with the glutamine residue; and

each P is independently an immunomodulating oligonucleotide; comprisingcontacting a compound of formula (B)

wherein Ab and Q are as defined for formula (A) above, and e is aninteger from 1 to 20, with one or more immunomodulating oligonucleotidesP, wherein each oligonucleotide P is independently an immunomodulatingoligonucleotide of formula (C) or formula (D), in the presence of atransglutaminase.

In some embodiments, the conjugate comprises one or more, two or more,three or more, four or more, five or more, six or more, seven or more,eight or more, nine or more, ten or more, or twenty or more Q-tagpeptides. In some embodiments, the conjugate comprises one, two, three,four, five, six, seven, eight, nine, ten, or twenty Q-tag peptides. Insome embodiments, the conjugate has 2 Q-tag peptides. In someembodiments, the conjugate comprises one or more, two or more, three ormore, four or more, five or more, six or more, seven or more, eight ormore, nine or more, ten or more, or twenty or more immunomodulatingoligonucleotides. In some embodiments, the conjugate comprises one, two,three, four, five, six, seven, eight, nine, ten, or twentyimmunomodulating oligonucleotides. In some embodiments, the conjugatehas one immunomodulating oligonucleotide.

In another aspect, the method comprises combining a compound of Formula(C) and an antibody of formula (B) comprising one or more glutamineresidues in the presence of a transglutaminase. In some embodiments, themethod comprises contacting a compound of Formula (D) and an antibody offormula (B) comprising one or more glutamine residues in the presence ofa transglutaminase. In some embodiments, the final concentration of thecompound of Formula (C) or Formula (D) is in the range of about 1-100μM. In some embodiments, the final concentration of the Q tag comprisingantibody is in the range of about 1-500 μM. In some embodiments, thefinal concentration of transglutaminase is in the range of about 1-500μM. In some embodiments, the final concentration of transglutaminase isin the range of about 1-50 μM, about 50-100 μM, about 100-150 μM, about150-200 μM, about 200-250 μM, about 250-300 μM, about 300-400 μM, about400-500 μM, about 100-125 μM, about 125-150 μM, about 150-175 μM, about175-200 μM, about 200-225 μM, about 225-250 μM, about 250-275 μM, about275-300 μM, about 300-325 μM or about 325-350 μM.

In some embodiments, the ratio of the Q tag comprising antibody and thecompound of Formula (C) or Formula (D) is in the range of about1:1-250:1, about 1:1-5:1, about 5:1-10:1, about 10:1-20:1, about20:1-30:1, about 30:1-40:1, about 40:1-50:1, about 50:1-75:1, about75:1-100:1, about 100:1-150:1, about 150:1-200:1, about 200:1-250:1,about 1:1-25:1, about 25:1-50:1, about 50:1-75:1, about 75:1-100:1 orabout 100:1-250:1 by weight. In some embodiments, the ratio of thecompound of Formula (C) or Formula (D) and the antibody is about 1:1,about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about8:1, about 9:1, about 10:1, about 11:1, about 12:1, about 13:1, about14:1, about 15:1, about 16:1, about 17:1, about 18:1 or about 20:1 bymolarity.

In some embodiments, the ratio of the Q tag comprising antibody andtransglutaminase is in the range of about 1:1-500:1, about 1:1-5:1,about 5:1-10:1, about 10:1-20:1, about 20:1-30:1, about 30:1-40:1, about40:1-50:1, about 50:1-75:1, about 75:1-100:1, about 100:1-150:1, about150:1-200:1, about 200:1-250:1, about 1:1-25:1, about 25:1-50:1, about50:1-75:1, about 75:1-100:1, about 100:1-150:1, about 150:1-200:1, about200:1-250:1, about 250:1-300:1, about 300:1-400:1 or about 400:1-500:1by weight. In some embodiments, the ratio of the peptide andtransglutaminase is about 15:1, about 16:1, about 17:1, about 18:1,about 20:1, about 21:1, about 22:1, about 23:1, about 24:1, about 25:1,about 26:1, about 27:1, about 28:1, about 29:1, about 30:1, about 31:1,about 32:1, about 33:1, about 34:1, about 35:1, about 36:1, about 37:1,about 38:1, about 39:1, about 40:1, about 41:1, about 42:1, about 43:1,about 44:1, about 45:1, about 46:1, about 47:1, about 48:1, about 49:1or about 50:1 by molarity.

In some embodiments, the ratio of Q tag: CpG: transglutaminase is about1:1.3:10. In some embodiments, the ratio of Q tag: CpG: transglutaminaseis about 1:1.5:10. In some embodiments, the ratio of Q tag: CpG:transglutaminase is about 1:1.3:15.

In some embodiments, the reaction is incubated at greater than 15° C.,greater than 20° C., greater than 25° C., greater than 30° C., greaterthan 35° C., greater than 40° C., greater than 45° C., or greater than50° C. In some embodiments, the reaction is incubated at about roomtemperature. In some embodiments, the reaction is incubated for at least10 minute, 20 minutes, 30 minutes, 45 minutes, 60 minutes, 2 hours, 3hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours,15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours, 50hours or 60 hours.

In some embodiments, the method described herein produces the compoundof Formula (A) at greater than about 5%, greater than about 10%, greaterthan about 15%, greater than about 20%, greater than about 25%, greaterthan about 30%, greater than about 35%, greater than about 40%, greaterthan about 45%, greater than about 50%, greater than about 60%, greaterthan about 70%, greater than about 80%, greater than about 90%, greaterthan about 95%, greater than about 97% or greater than about 99% ascompared to the peptide.

In some embodiments, the pH of the reaction is in the range of about4-10. In some embodiments, the pH of the reaction is in the range ofabout 4-6, about 6-8 or about 8-10. In some embodiments, the pH of thereaction is in the range of about 7-8.

In another aspect, reactions useful for attaching a linking moiety to anoligonucleotide are known in the art, including, but not limited toHisgen cycloaddition (metal-catalyzed or metal-free) between an azidoand an alkyne-based conjugating group (e.g., optionally substitutedC₆₋₁₆ heterocyclylene containing an endocyclic carbon-carbon triple bondor optionally substituted C₈₋₁₆ cycloalkynyl) to form a triazole moiety;the Diels-Alder reaction between a dienophile and a diene/hetero-diene;bond formation via pericyclic reactions such as the ene reaction; amideor thioamide bond formation; sulfonamide bond formation (e.g., withazido compounds); alcohol or phenol alkylation (e.g., Williamsonalkylation), condensation reactions to form oxime, hydrazone, orsemicarbazide group; conjugate addition reactions by nucleophiles (e.g.,amines and thiols); disulfide bond formation; and nucleophilicsubstitution (e.g., by an amine, thiol, or hydroxyl nucleophile) at acarbonyl (e.g., at an activated carboxylic acid ester, such aspentafluorophenyl (PFP) ester or tetrafluorophenyl (TFP) ester) or at anelectrophilic arene (e.g., SNAr at an oligofluorinated arene, afluorobenzonitrile group, or fluoronitrobenzene group).

In certain embodiments, the attachment reaction is a dipolarcycloaddition, and the conjugation moiety includes azido, optionallysubstituted C₆₋₁₆ heterocyclylene containing an endocyclic carbon-carbontriple bond, or optionally substituted C₈₋₁₆ cycloalkynyl. Thecomplementary reactive group and the conjugating group are selected fortheir mutual complementarity. For example, an azide is used in one ofthe conjugating group and the complementary reactive group, while analkyne is used in the other of the conjugating group and thecomplementary reactive group.

Attachment of Linking Moiety to the Oligonucleotide

A linking moiety can be attached to an oligonucleotide by forming a bondbetween a attaching group in the oligonucleotide and a complementaryreactive group bonded to the linking moiety. In certain embodiments, thelinking moiety, is modified to include a complementary reactive group.Methods of introducing such complementary reactive groups into a linkingmoiety is known in the art.

In certain embodiments, the complementary reactive group is optionallysubstituted C₂₋₁₂ alkynyl, optionally substituted N-protected amino,azido, N-maleimido, S-protected thiol,

or a N-protected moiety thereof,

optionally substituted C₆₋₁₆ heterocyclyl containing an endocycliccarbon-carbon triple bond (e.g.,

1,2,4,5-tetrazine group (e.g.,

optionally substituted C₈₋₁₆ cycloalkynyl (e.g.,

—NHR^(N1), optionally substituted C₄₋₈ strained cycloalkenyl (e.g.,trans-cyclooctenyl or norbornenyl), or optionally substituted C₁₋₁₆alkyl containing —COOR¹² or —CHO;wherein:

R^(N1) is H, N-protecting group, or optionally substituted C₁₋₆ alkyl;

each R¹² is independently H, optionally substituted C₁₋₆ alkyl, orO-protecting group (e.g., a carboxyl protecting group); and

R¹³ is halogen (e.g., F).

In certain embodiments, the complementary reactive group is protecteduntil the conjugation reaction. For example, a complementary reactivegroup that is protected can include —COOR^(PGO) or —NHR^(PGN), whereR^(PGO) is an O-protecting group (e.g., a carboxyl protecting group),and R^(PGN) is an N-protecting group.

VI. Pharmaceutical Compositions

The compounds and conjugates of the present invention, such as theconjugates comprising structures of formula (A), antibodies of formula(B), and immunomodulating oligonucleotides of formulae (C), (C′), (C″),(D), (D′) and (D″), or a pharmaceutically acceptable salt of any of theforegoing, or any subgroup thereof may be formulated into variouspharmaceutical forms for administration purposes. As appropriatecompositions there may be cited all compositions usually employed forsystemically administering drugs. To prepare the pharmaceuticalcompositions of this invention, an effective amount of the particularcompound, optionally in addition salt form, as the active ingredient iscombined in admixture with a pharmaceutically acceptable carrier, whichcarrier may take a wide variety of forms depending on the form ofpreparation desired for administration. These pharmaceuticalcompositions are desirable in unitary dosage form suitable,particularly, for administration orally, rectally, percutaneously, or byparenteral injection. For example, in preparing the compositions in oraldosage form, any of the usual pharmaceutical media may be employed suchas, for example, water, glycols, oils, alcohols and the like in the caseof oral liquid preparations such as suspensions, syrups, elixirs,emulsions and solutions; or solid carriers such as starches, sugars,kaolin, lubricants, binders, disintegrating agents and the like in thecase of powders, pills, capsules, and tablets. Because of their ease inadministration, tablets and capsules represent the most advantageousoral dosage unit forms, in which case solid pharmaceutical carriers areemployed. For parenteral compositions, the carrier will usually comprisesterile water, at least in large part, though other ingredients, forexample, to aid solubility, may be included. Injectable solutions, forexample, may be prepared in which the carrier comprises saline solution,glucose solution or a mixture of saline and glucose solution. Injectablesuspensions may also be prepared in which case appropriate liquidcarriers, suspending agents and the like may be employed. Also includedare solid form preparations intended to be converted, shortly beforeuse, to liquid form preparations. In the compositions suitable forpercutaneous administration, the carrier optionally comprises apenetration enhancing agent and/or a suitable wetting agent, optionallycombined with suitable additives of any nature in minor proportions,which additives do not introduce a significant deleterious effect on theskin. The compounds of the present invention may also be administeredvia oral inhalation or insufflation in the form of a solution, asuspension or a dry powder using any art-known delivery system.

It is especially advantageous to formulate the aforementionedpharmaceutical compositions in unit dosage form for ease ofadministration and uniformity of dosage. Unit dosage form as used hereinrefers to physically discrete units suitable as unitary dosages, eachunit containing a predetermined quantity of active ingredient calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier. Examples of such unit dosage forms aretablets (including scored or coated tablets), capsules, pills,suppositories, powder packets, wafers, injectable solutions orsuspensions and the like, and segregated multiples thereof.

Administration can be, but is not limited to, intravenous,intraarterial, subcutaneous, intraperitoneal, subdermal (e.g., via animplanted device), and intraparenchymal administration. In someembodiments, the pharmaceutical compositions described herein areadministered by subcutaneous injection.

The pharmaceutical compositions including a conjugate described hereincan be delivered to a cell, group of cells, tumor, tissue, or subjectusing delivery technologies known in the art. In general, any suitablemethod recognized in the art for delivering a nucleic acid-proteinconjugate (in vitro or in vivo) can be adapted for use with a hereindescribed compositions. For example, delivery can be by localadministration, (e.g., direct injection, implantation, or topicaladministering), systemic administration, or subcutaneous, intravenous,intraperitoneal, or parenteral routes, including intracranial (e.g.,intraventricular, intraparenchymal and intrathecal), intramuscular,transdermal, airway (aerosol), nasal, oral, rectal, or topical(including buccal and sublingual) administration. In certainembodiments, the compositions are administered by subcutaneous orintravenous infusion or injection.

Accordingly, in some embodiments, the herein described pharmaceuticalcompositions may comprise one or more pharmaceutically acceptableexcipients. In some embodiments, the pharmaceutical compositionsdescribed herein can be formulated for administration to a subject.

As used herein, a pharmaceutical composition or medicament includes apharmacologically effective amount of at least one of the describedtherapeutic compounds or conjugates and one or more pharmaceuticallyacceptable excipients. Pharmaceutically acceptable excipients(excipients) are substances other than the Active Pharmaceuticalingredient (API, therapeutic product) that are intentionally included inthe drug delivery system. Excipients do not exert or are not intended toexert a therapeutic effect at the intended dosage. Excipients may act toa) aid in processing of the drug delivery system during manufacture, b)protect, support or enhance stability, bioavailability or patientacceptability of the API, c) assist in product identification, and/or d)enhance any other attribute of the overall safety, effectiveness, ofdelivery of the API during storage or use. A pharmaceutically acceptableexcipient may or may not be an inert substance.

Excipients include, but are not limited to: absorption enhancers,anti-adherents, anti-foaming agents, anti-oxidants, binders, bufferingagents, carriers, coating agents, colors, delivery enhancers, deliverypolymers, dextran, dextrose, diluents, disintegrants, emulsifiers,extenders, fillers, flavors, glidants, humectants, lubricants, oils,polymers, preservatives, saline, salts, solvents, sugars, suspendingagents, sustained release matrices, sweeteners, thickening agents,tonicity agents, vehicles, water-repelling agents, and wetting agents.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline. It should bestable under the conditions of manufacture and storage and should bepreserved against the contaminating action of microorganisms such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyethylene glycol), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.In many cases, it will be preferable to include isotonic agents, forexample, sugars, polyalcohols such as mannitol, sorbitol, and sodiumchloride in the composition. Prolonged absorption of the injectablecompositions can be brought about by including in the composition anagent which delays absorption, for example, aluminum monostearate andgelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfilter sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation include vacuumdrying and freeze-drying which yields a powder of the active ingredientplus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Formulations suitable for intra-articular administration can be in theform of a sterile aqueous preparation of the drug that can be inmicrocrystalline form, for example, in the form of an aqueousmicrocrystalline suspension. Liposomal formulations or biodegradablepolymer systems can also be used to present the drug for bothintra-articular and ophthalmic administration.

The active compounds can be prepared with carriers that will protect thecompound against rapid elimination from the body, such as a controlledrelease formulation, including implants and microencapsulated deliverysystems. Biodegradable, biocompatible polymers can be used, such asethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Methods for preparation of suchformulations will be apparent to those skilled in the art. Liposomalsuspensions can also be used as pharmaceutically acceptable carriers.These can be prepared according to methods known to those skilled in theart, for example, as described in U.S. Pat. No. 4,522,811.

The compound or conjugate can be formulated in compositions in dosageunit form for ease of administration and uniformity of dosage. Dosageunit form refers to physically discrete units suited as unitary dosagesfor the subject to be treated; each unit containing a predeterminedquantity of active compound calculated to produce the desiredtherapeutic effect in association with the required pharmaceuticalcarrier. The specification for the dosage unit forms of the disclosureare dictated by and directly dependent on the unique characteristics ofthe active compound and the therapeutic effect to be achieved, and thelimitations inherent in the art of compounding such an active compoundfor the treatment of individuals.

A pharmaceutical composition can contain other additional componentscommonly found in pharmaceutical compositions. Such additionalcomponents include, but are not limited to: anti-pruritics, astringents,local anesthetics, or anti-inflammatory agents (e.g., antihistamine,diphenhydramine, etc.).

Generally, an effective amount of an active compound will be in therange of from about 0.1 to about 100 mg/kg of body weight/day, e.g.,from about 1.0 to about 50 mg/kg of body weight/day. In someembodiments, an effective amount of an active compound will be in therange of from about 0.25 to about 5 mg/kg of body weight per dose. Insome embodiments, an effective amount of an active compound will be inthe range of 25-400 mg per 1-18 weeks or 1-6 months. In someembodiments, an effective amount of an active compound will be in therange of 50-125 mg per 4 weeks or per one month. In some embodiments, aneffective amount of an active ingredient will be in the range of fromabout 0.5 to about 3 mg/kg of body weight per dose. In some embodiments,an effective amount of an active ingredient will be in the range of fromabout 25-400 mg per dose. In some embodiments, an effective amount of anactive ingredient will be in the range of from about 50-125 mg per dose.The amount administered will also likely depend on such variables as theoverall health status of the patient, the relative biological efficacyof the compound delivered, the formulation of the drug, the presence andtypes of excipients in the formulation, and the route of administration.Also, it is to be understood that the initial dosage administered can beincreased beyond the above upper level in order to rapidly achieve thedesired blood-level or tissue level, or the initial dosage can besmaller than the optimum.

For treatment of disease or for formation of a medicament or compositionfor treatment of a disease, the pharmaceutical compositions describedherein including a compound or conjugate can be combined with anexcipient or with a second therapeutic agent or treatment including, butnot limited to: a second or other conjugates, a small molecule drug, anantibody, an antibody fragment, and/or a vaccine.

The described compounds or conjugates, when added to pharmaceuticallyacceptable excipients or adjuvants, can be packaged into kits,containers, packs, or dispensers. The pharmaceutical compositionsdescribed herein may be packaged in pre-filled syringes or vials.

VII. Kits

Also provided herein is a kit comprising a conjugate as described above.

In another aspect, the kit further comprises a package insert including,without limitation, appropriate instructions for preparation andadministration of the formulation, side effects of the formulation, andany other relevant information. The instructions may be in any suitableformat, including, but not limited to, printed matter, videotape,computer readable disk, optical disc or directions to internet-basedinstructions.

In another aspect, kits for treating an individual who suffers from oris susceptible to the conditions described herein are provided,comprising a first container comprising a dosage amount of a compositionor formulation as disclosed herein, and a package insert for use. Thecontainer may be any of those known in the art and appropriate forstorage and delivery of intravenous formulation. In certain embodiments,the kit further comprises a second container comprising apharmaceutically acceptable carrier, diluent, adjuvant, etc. forpreparation of the formulation to be administered to the individual.

In another aspect, kits may also be provided that contain sufficientdosages of the compositions described herein (including pharmaceuticalcompositions thereof) to provide effective treatment for an individualfor an extended period, such as 1-3 days, 1-5 days, a week, 2 weeks, 3,weeks, 4 weeks, 6 weeks, 8 weeks, 1 cycle, 2 cycles, 3 cycles, 4 cycles,5 cycles, 6 cycles, 7 cycles, 8 cycles or more.

In some embodiments, the kits may also include multiple doses and may bepackaged in quantities sufficient for storage and use in pharmacies, forexample, hospital pharmacies and compounding pharmacies. In certainembodiments the kits may include a dosage amount of at least onecomposition as disclosed herein.

VIII. Methods of Treatment

Also provided herein are methods for treating a disease or disorder in asubject comprising administering an effective amount of a compound orconjugate described herein to the subject in need thereof. Also providedherein are uses of a compound or conjugate described herein in thepreparation of a medicament for treating a patient in need of treatmentwith the oligonucleotide in the conjugate. Also provided are compoundsor conjugates as described herein for treating a disease or disorder ina subject in need of the treatment with the oligonucleotide in thecompounds or conjugates. Also provided are compounds or conjugates asdescribed herein for treating a patient comprising administering aneffective amount of the compound or conjugate to the patient. In someembodiments, the subject has or at the risk of developing cancer. Insome embodiments, the disease or disorder is a viral infection. In someembodiments, the disease or disorder is an immunodeficiency, e.g., inwhich immune activation may be favorable. In some embodiments, thedisease or disorder is an autoimmune and/or inflammatory disease ordisorder, e.g., in which immune suppression and/or modulation may befavorable.

In some embodiments of the methods of treating cancer as describedherein, the cancer being treated with the methods disclosed herein is asolid tumor. In some embodiments, the cancer being treated with themethods disclosed herein is a liquid tumor. In some embodiments, thecancer being treated with the methods disclosed herein is a solid tumor.In particular embodiments, the cancer being treated with the methodsdisclosed herein is breast cancer, colorectal cancer, lung cancer, headand neck cancer, melanoma, lymphoma, or leukemia. In some embodiments,cancers include, but are not limited to, B cell cancer, e.g., multiplemyeloma, Waldenström's macroglobulinemia, the heavy chain diseases, suchas, for example, alpha chain disease, gamma chain disease, and mu chaindisease, benign monoclonal qammopathy, and immunocytic amyloidosis,melanomas, breast cancer, lung cancer, bronchus cancer, colorectalcancer, prostate cancer, pancreatic cancer, stomach cancer, ovariancancer, urinary bladder cancer, brain or central nervous system cancer,peripheral nervous system cancer, esophageal cancer, cervical cancer,uterine or endometrial cancer, cancer of the oral cavity or pharynx,liver cancer, kidney cancer, testicular cancer, biliary tract cancer,small bowel or appendix cancer, salivary gland cancer, thyroid glandcancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, cancer ofhematologic tissues, and the like. Other non-limiting examples of typesof cancers applicable to the methods encompassed by the presentinvention include human sarcomas and carcinomas, e.g., fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, colorectal cancer,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, liver cancer, choriocarcinoma, sominoma, embryonal carcinoma,Wilms' tumor, cervical cancer, bone cancer, brain tumor, testicularcancer, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma,retinoblastoma; leukemias, e.g., acute lymphocytic leukemia and acutemyelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic,monocytic and erythroleukemia); chronic leukemia (chronic myelocytic(granulocytic) leukemia and chronic lymphocytic leukemia); andpolycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin'sdisease), multiple myeloma, Waldenstrom's macroglobulinemia, and heavychain disease. In some embodiments, cancers are epithlelial in natureand include but are not limited to, bladder cancer, breast cancer,cervical cancer, colon cancer, gynecologic cancers, renal cancer,laryngeal cancer, lung cancer, oral cancer, head and neck cancer,ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer. Inother embodiments, the cancer is breast cancer, prostate cancer, lungcancer, or colon cancer. In still other embodiments, the epithelialcancer is non-small-cell lung cancer, nonpapillary renal cell carcinoma,cervical carcinoma, ovarian carcinoma (e.g., serous ovarian carcinoma),or breast carcinoma. The epithelial cancers may be characterized invarious other ways including, but not limited to, serous, endometrioid,mucinous, clear cell, Brenner, or undifferentiated. In particularembodiments, the cancer being treated with the methods disclosed hereinis selected from the list consisting of mantle cell cymphoma (MCL),diffuse large B-cell lymphoma (DLBCL), Burkitts lymphoma, multiplemelanoma (MM), chronic lymphocytic leukemia (CLL), acute myeloidleukemia (AML), small lymphocytic lymphoma (SLL), hairy cell leukemia(HCL), lymphoplasmacytic lymphoma (LPL), skeletal muscle lymphoma (SML),splenic marginal zone lymphoma (SMZL), follicle center lymphoma (FCL),colorectal cancer, non-small cell lung cancer (NSCLC), head and neckcancer, breast cancer, pancreatic cancer, glioblastoma (GBM), prostatecancer, esophageal cancer, renal cell carcinoma, hepatic carcinoma,bladder cancer and gastric carcinoma.

In some embodiments, provided herein methods for treating a disease ordisorder in a subject comprising administering an effective amount of aCpG-Ab immunoconjugate described herein to the subject in need thereof,wherein the CpG-Ab immunoconjugate binds to CD22, such as a CpG-Abimmunoconjugate comprising an anti-CD22 antibody or antigen bindingfragment thereof, and where the disease or disorder treated is a cancercharacterized by tumor infiltrating B cells. In some embodiments, suchcancers include head and neck squamous cell carcinoma (HNSCC),non-small-cell lung carcinoma (NSCLC), renal cell carcinoma (RCC),gastric cancer, hepatocellular carcinoma (HCC), esophageal cancer,cervical cancer, Merkle cell carcinoma, endometrial cancer, acutelymphoblastic leukemia (ALL), hairy cell leukemia, and diffuse large Bcell lymphoma (DLBCL). In some embodiments, such cancers include ovariancancer, pancreatic cancer, melanoma, cutaneous melanoma, sarcoma,colorectal cancer, breast cancer, cervical squamous cell carcinoma,small cell lung cancer (SCLC), cutaneous squamous cell carcinoma, andurothelial carcinoma. In some embodiments, the CpG-Ab immunoconjugate islisted in one of Tables 2 and 9-12.

In some embodiments, the methods of treatment include administration ofa CpG-Ab immunoconjugate that binds to CD22 expressed on the surface ofa B cell and the treatment results in one or more of B cell activation,B cell differentiation, increased in T cell effector function, T cellproliferation, secretion of cytokines and chemokines, induction ofimmunoglobulin secretion and modulation in suppressive myeloid cellswithin the tumor microenvironment in the treated subject. In someembodiments, the methods of treatment include administration of a CpG-Abimmunoconjugate that binds to CD22 expressed on the surface of a B celland the treatment results a memory immune response. In some embodiments,such treatment results in anti-tumor activity through both innate andadaptive immune responses.

In some embodiments, the methods of treatment include administration ofa CpG-Ab immunoconjugate that binds to HER2 present on a tumor cell andthe treatment results in the killing of or impairment of tumor cell(s)such that the volume, size and/or growth of the tumor is reduced orinhibited. In some embodiments, provided herein are methods for treatinga disease or disorder in a subject comprising administering an effectiveamount of a CpG-Ab immunoconjugate described herein to the subject inneed thereof, wherein the CpG-Ab immunoconjugate binds to HER2, such asa CpG-Ab immunoconjugate comprising an anti-HER2 antibody or antigenbinding fragment thereof, and where the disease or disorder treated is acancer characterized by HER2-expressing tumor cells. In someembodiments, the disease or disorder treated is a cancer where tumorcells over-express HER2. In some embodiments, the cancer is breastcancer, urothelial cancer or gastric cancer.

In some embodiments, the cancer being treated with the methods disclosedherein is resistant to at least one immunotherapy. In some embodiments,the cancer being treated with the methods disclosed herein is resistantto at least one cancer therapy selected from the group consisting ofchemotherapy, radiation, targeted therapy, vaccine therapy, and CAR-Ttherapy. In some embodiments, the method of treating cancer comprisesco-administering to a subject having cancer (i) a therapeuticallyeffective amount of the CpG-containing immunostimulating polynucleotideor the CpG-antibody immunoconjugate; and (ii) the immunotherapeuticagent which the cancer being treated has shown to resist or not torespond, when the cancer is treated with the immunotherapeutic agentalone.

In particular embodiments, the cancer being treated with the methodsprovided herein has been shown to not to respond to a treatment with animmune checkpoint modulator. In particular embodiments, the immunecheckpoint modulator is an inhibitor of PD-1. In particular embodiments,the immune checkpoint modulator is an inhibitor of PD-L1. In someembodiments, the method of treating cancer comprises co-administering toa subject having cancer (i) a therapeutically effective amount of theCpG-containing immunostimulating polynucleotide or the CpG-Abimmunoconjugate; and (ii) a therapeutically effective amount of theinhibitor of PD-1. In some embodiments, the method of treating cancercomprises co-administering to a subject having cancer (i) atherapeutically effective amount of the CpG-containing immunostimulatingpolynucleotide or the CpG-Ab immunoconjugate; and (ii) a therapeuticallyeffective amount of the inhibitor of PD-L1. In particular, in someembodiments, the inhibitor of PD-1 is an anti-PD-1 antibody or anantigen-binding fragment thereof. In some embodiments, the inhibitor ofPD-L1 is an anti-PD-L1 antibody or an antigen-binding fragment thereof.In some embodiments, the treatment is directed to a subject that doesnot respond to or is resistant to a PD-1 or PD-L1 inhibitor and suchsubject is treated with a CpG-Ab immunoconjugate that binds CD22, suchas a CpG-Ab immunoconjugate comprising an anti-CD22 antibody or antigenbinding fragment thereof.

In certain aspects, provided herein are methods of preventing cancer ina subject susceptible of developing cancer, comprising administering tothe subject a therapeutically effective amount of a TLR agonist asdescribed herein. In some embodiments, the method comprisingadministering to the subject a therapeutically effective amount of aCpG-containing immunostimulating polynucleotide or a CpG-Abimmunoconjugate described herein. In particular embodiments, the CpG-Abimmunoconjugate targets an immune cell as described herein. Inparticular embodiments, the CpG-Ab immunoconjugate targets aTLR-expressing cell as described herein. In particular embodiments, theCpG-Ab immunoconjugate specifically binds to an antigen associated withan immune cell as described herein. In particular embodiments, theCpG-Ab immunoconjugate specifically binds to an antigen associated withan immune cell does not bind to a tumor-associated antigen of the cancerbeing prevented. In particular embodiments, the CpG-Ab immunoconjugatespecifically binds to an antigen associated with a TLR-expressing cellas described herein. In particular embodiments, the CpG-Abimmunoconjugate specifically binds to an antigen associated with aTLR-expressing cell and does not bind to a tumor-associated antigen ofthe cancer being prevented. In particular embodiments, the CpG-Abimmunoconjugate specifically binds to a tumor-associated antigen of thecancer being prevented as described herein, e.g., an antigen expressedon a tumor cell surface. In particular embodiments, a tumor-associatedantigen of the cancer being prevented is also associated with an immunecell or a TLR-expressing cell. In particular embodiments, the CpG-Abimmunoconjugate does not specifically bind to an antigen selected fromCD19, CD20, CD22, STAT3, exportin 7, Her2, Src, EGFR, CD52, CXCR-4, andMuc-1.

In some embodiments, the methods of preventing cancer further comprisesadministering to a subject susceptible to developing cancer (i) atherapeutically effective amount of a CpG-Ab immunoconjugate and (ii) atumor-associated antigen of the cancer being prevented. In someembodiments, the tumor-associated antigen is not conjugated to theCpG-Ab immunoconjugate. In particular embodiments, the tumor-associatedantigen is formulated as a cancer vaccine. In particular embodiments,the CpG-Ab immunoconjugate is formulated as an adjuvant of the cancervaccine.

In some embodiments, the cancer being prevented or treated using themethods provided herein is an episode of cancer recurrence in a subjectwho is in partial or complete remission of a prior cancer. In particularembodiments, the prior cancer is a liquid cancer and the recurrentcancer being prevented or treated is a liquid cancer. In particularembodiments, the prior cancer is a solid cancer and the recurrent cancerbeing prevented or treated is a solid cancer. In particular embodiments,the prior cancer is a liquid cancer and the recurrent cancer beingprevented or treated is a solid cancer. In particular embodiments, theprior cancer is a solid cancer and the recurrent cancer being preventedor treated is a liquid cancer.

In some embodiments, the cancer being prevented or treated using themethods provided herein is first episode of cancer recurrence in thesubject after the subject showed partial or complete remission. In someembodiments, the cancer being prevented or treated using the methodsprovided herein is second episode of cancer recurrence in the subjectafter the subject showed partial or complete remission. In someembodiments, the cancer being prevented or treated using the methodsprovided herein is third episode of cancer recurrence in the subjectafter the subject showed partial or complete remission. In someembodiments, the cancer being prevented or treated using the methodsprovided herein is an episode of cancer recurrence subsequent to thethird episode of cancer recurrence in the subject after the subjectshowed partial or complete remission.

In certain aspects, provided herein are methods of inducing an adaptiveimmune response in a subject in need thereof, wherein method comprisesadministering to the subject an therapeutically effective amount of aTLR agonist as described herein. In particular embodiments, the methodof inducing an adaptive immune response comprises administering to thesubject in need thereof a therapeutically effective amount of aCpG-containing immunostimulating polynucleotide or a CpG-Abimmunoconjugate described herein. In particular embodiments, the CpG-Abimmunoconjugate targets a normal immune cell as described herein. Inparticular embodiments, the CpG-Ab immunoconjugate targets aTLR-expressing cell as described herein. In particular embodiments, theCpG-Ab immunoconjugate targets a diseased cell selected from a cancercell or a pathogen infected cell. In particular embodiments, the CpG-Abimmunoconjugate specifically binds to an antigen associated with anormal immune cell as described herein. In particular embodiments, theCpG-Ab immunoconjugate specifically binds to an antigen associated witha normal immune cell does not bind to a disease antigen. In particularembodiments, the CpG-Ab immunoconjugate specifically binds to an antigenassociated with a TLR-expressing cell as described herein. In particularembodiments, the CpG-Ab immunoconjugate specifically binds to an antigenassociated with a TLR-expressing cell does not bind to a diseaseantigen. In particular embodiments, the CpG-Ab immunoconjugatespecifically binds to a disease antigen as described herein. Inparticular embodiments, the diseased antigen is also associated with anormal immune cell or a TLR-expressing cell. In particular embodiments,the diseased antigen is a tumor-associated antigen or a pathogenicantigen. In particular embodiments, the CpG-Ab immunoconjugate does notspecifically bind to an antigen selected from CD19, CD20, CD22, STAT3,exportin 7, Her2, Src, EGFR, CD52, CXCR-4, and Muc-1. In someembodiments, the antibody or conjugate specifically binds an antigenexpressed by a cancer or cancer-associated stroma.

In some embodiments of the methods and uses described herein, theCpG-containing immunostimulating polynucleotide is administered to asubject in need thereof at a dosage that is sufficient for activatingthe TLR9-mediated signaling pathway in the subject. In some embodiments,the CpG-Ab immunoconjugate is administered to a subject in need thereofat a dosage that is sufficient for activating the TLR9 mediatedsignaling pathway in a cell population targeted by the CpG-Abimmunoconjugate. As described herein, in some embodiments, the cellpopulation targeted by the CpG-Ab immunoconjugate expresses TLR9. Insome embodiments, the cell population targeted by the CpG-Abimmunoconjugate can express the TLR9 on the cell surface of the targetedcell, on the endosomal membrane of the targeted cell, or both on thecell surface and on the endosomal membrane of the targeted cell.

Particularly, in some embodiments of the methods and uses describedherein, the CpG-containing immunostimulating polynucleotide isadministered to a subject in need thereof at a dosage that is effectivefor inducing one or more of effects selected from (a) specificallybinding to a TLR9 receptor by the CpG-containing immunostimulatingpolynucleotide on a targeted cell; (b) efficient internalization of theCpG-Ab immunoconjugate or the CpG-containing immunostimulatingpolynucleotide portion thereof by a targeted cell; (c) activating one ormore signaling pathways in the targeted cell; (d) inducing secretion ofone or more inflammatory cytokines by the targeted cell; (e) suppressingsecretion of one or more inflammatory cytokines by the targeted cell;(f) upregulating expression of one or more genes of the targeted cell;(g) suppressing expression of one or more genes of the targeted cell;(h) activating targeted normal immune cells, (i) inducing an immuneresponse that results in the elimination of disease, e.g. cancer cells,(j) inducing apoptosis of a targeted cancer cell, and (k) inducingnecrosis of targeted cancer cell.

Particularly, in some embodiments of the methods and uses describedherein, wherein upon administration of the CpG-Ab immunoconjugate, theCpG-containing immunostimulating polynucleotide specifically binds to aTLR9 receptor of the targeted cell. Particularly, in some embodiments,binding of CpG-Ab immunoconjugate to an antigen associated with atargeted cell facilitates specific binding of the CpG-containingimmunostimulating polynucleotide to a TLR9 receptor. In someembodiments, the target antigen of the CpG-Ab immunoconjugate is locatednear the TLR9 receptor. In particular embodiments, both the targetantigen and the TLR9 receptor locate on the cell membrane of thetargeted cell. In particular embodiments, both the target antigen andthe TLR9 receptor locate on an intracellular membrane of the targetedcell. In particular embodiments, both the target antigen and the TLR9receptor locate on the endosomal or phagosomal membrane of the targetedcell. In some embodiments, the target antigen locates on the cellmembrane and facilitates internalization of the CpG-Ab immunoconjugateinto the cytosol upon binding to the CpG-Ab immunoconjugate.

Particularly, in some embodiments of the methods and uses describedherein, the method comprises administering to a subject in need thereofa therapeutically effective amount of a CpG-Ab immunoconjugate targetinga normal immune cell, wherein upon administration of the CpG-Abimmunoconjugate, one or more immunogenic signaling pathways in thetargeted cell are activated. In particular embodiments, the activatedsignaling pathways are one or more selected from the nuclear factor(NF)-κB signaling pathway, the c-Jun N-terminal kinase (INK) signalingpathway, the AP1 signaling pathway, the IRF3/7 pathway, and the p38mitogen-activated protein kinase (MAPK) signaling pathway. Theactivation of a cellular signaling pathway can be detected using methodsknown in the art, such as but not limited to, detecting the presence ofa molecular marker of which the expression is specifically induced uponactivation of the signaling pathway of interest.

Particularly, in some embodiments of the methods and uses describedherein, the method comprises administering to a subject in need thereofa therapeutically effective amount of a CpG-Ab immunoconjugate targetinga normal immune cell, wherein upon administration of the CpG-Abimmunoconjugate, secretion of one or more inflammatory cytokines isinduced. In particular embodiments, the one or more inflammatorycytokines are selected from type I interferon (IFN), interleukin (IL)-6,IL10, IL-12, IL-18, and tumor necrosis factor (TNF).

Particularly, in some embodiments of the methods and uses describedherein, the method comprises administering to a subject in need thereofa therapeutically effective amount of a CpG-Ab immunoconjugate targetinga normal immune cell, wherein upon administration of the CpG-Abimmunoconjugate, expression of one or more additional proteins areupregulated. In particular embodiments, the upregulated proteins are oneor more selected from antigen presenting molecules (e.g., MHC class Iand II), cytokine receptors (e.g., IL-6 receptors, IL-10 receptors,IL-12 receptors, TNF-α receptor, TNF-β receptor, IFN-α receptor, IFN-βreceptor, IFN-γ), chemokine receptors (e.g., chemokine receptor 7),costimulatory molecules (e.g., CD3, CD28, CD27, CD30, CD40, CD69,CD80/B7-1, CD86/B7-2, CD134/OX-40, OX-40L, CD137/4-1BB, 4-1BBL,CD278/ICOS, B7-H3, B7h/B7RP-1, LIGHT etc.), HLA-DR and T cell maturationregulatory proteins (e.g., indoleamine 2,3-dioxygenase).

Particularly, in some embodiments of the methods and uses describedherein, the method comprises administering to a subject in need thereofa therapeutically effective amount of a CpG-Ab immunoconjugate targetinga normal immune cell, wherein upon administration of the CpG-Abimmunoconjugate, proliferation, differentiation, maturation and/orsurvival of one or more populations of normal immune cells areincreased. In particular embodiments, the one or more increasedpopulations of normal immune cells are selected from CD4+ T cells, CD8+T cells, natural killer cells, T helper cells, B cells, and myeloidcells (including mDCs and pDCs). in some embodiments of the methods anduses described herein, the method comprises administering to a subjectin need thereof a therapeutically effective amount of a CpG-Abimmunoconjugate targeting a normal immune cell, wherein uponadministration of the CpG-Ab immunoconjugate, proliferation,differentiation, maturation and/or survival of one or more populationsof normal immune cells are reduced. In particular embodiments, the oneor more reduced populations of normal immune cells is selected fromB-reg cells, T-reg cells, and MDSCs.

In particular embodiments, upon administration of the CpG-Abimmunoconjugate, antigen presentation activities are increased in APCsin the subject. In some embodiments, the APC is selected from B cells,monocytes, dendritic cells, and Langerhans cells, keratinocytes,endothelial cells, astrocytes, fibroblasts, and oligodendrocytes. Inparticular embodiments, the APC is B cells. In particular embodiments,the APC is dendritic cells. In particular embodiments, the APC ismacrophage. In some embodiments, the dendritic cell is pDC. Inparticular embodiments, the increased antigen presentation activitieslead to more efficient presentation of a tumor-associated antigen by theactivated APCs.

In particular embodiments, upon administration of the CpG-Abimmunoconjugate, antigen-specific CD4+ T cell mediated immunity againstone or more tumor-associated antigen of the cancer being treated orprevented is increased. In particular embodiments, upon administrationof the CpG-Ab immunoconjugate, tumor infiltration by CD4+ T cell isincreased. In particular embodiments, upon administration of the CpG-Abimmunoconjugate, antigen-specific CD8+ T cell mediated immunity againstone or more tumor-associated antigen of the cancer being treated orprevented is increased is increased. In particular embodiments, uponadministration of the CpG-Ab immunoconjugate, tumor infiltration by CD8+T cell is increased. In particular embodiments, upon administration ofthe CpG-Ab immunoconjugate, B cell secretion of immunoglobulinspecifically against one or more tumor-associated antigen of the cancerbeing treated or prevented is increased is increased.

Particularly, in some embodiments of the methods and uses describedherein, the method comprises administering to a subject in need thereof,a therapeutically effective amount of a CpG-Ab immunoconjugate targetinga diseased cell, wherein upon administration of the CpG-Abimmunoconjugate, one or more apoptotic signaling pathways are inducedtrigger apoptosis of the targeted diseased cell. In some embodiments,the diseased cell is a cancer cell.

In some embodiments of the methods and uses described herein, the CpG-Abimmunoconjugate is administered to a subject in need thereof in anamount that is not effective for activating the complement system in thesubject. In some embodiments, the CpG-containing immunostimulatingpolynucleotide is administered to a subject in need thereof in an amountthat is not effective to activate complement C1 in the subject. In someembodiments, the CpG-containing immunostimulating polynucleotide isadministered to a subject in need thereof in an amount that is noteffective to activate complement C3 in the subject. Complementactivation can be detected using methods known in the art. In someembodiments, the CpG-Ab immunoconjugate is administered to a subject inneed thereof in an amount that is not effective for the antibody portionof the CpG-Ab immunoconjugate to induce antibody-dependent cell-mediatedcytotoxicity in the subject.

As described herein, therapeutic agents, conjugates or compositionscomprising the CpG-containing polynucleotides can be used in combinationwith at least one additional therapeutic agent for preventing ortreating cancer. In some embodiments, such combination therapy exhibitsa synergistic therapeutic effect that is better than the separate effectof either therapeutic agent alone. In some embodiments, such combinationtherapies exhibit a synergistic therapeutic effect that is better thanthe sum of the separate effects of the therapeutic agents alone.

Accordingly, in certain aspects, provided herein are methods forpreventing or treating cancer using the CpG-containing immunostimulatingpolynucleotide in combination with at least one additional cancertherapeutic agent. Such methods comprising administering to a subject inneed thereof (i) a therapeutically effective amount of theCpG-containing immunostimulating polynucleotide, and (ii) atherapeutically effective amount of at least one additional cancertherapeutic agents. In particular embodiments, the CpG-containingimmunostimulating polynucleotide is administered as a free-standingpolynucleotide. In particular embodiments, the CpG-containingimmunostimulating polynucleotide is administered as a CpG-Abimmunoconjugate. In particular embodiments, the CpG-containingimmunostimulating polynucleotide and the additional therapeutic agentsare formulated in the same composition. In other embodiments,CpG-containing immunostimulating polynucleotide and the additionaltherapeutic agents are formulated in the separate compositions.

In some embodiments, the at least one additional cancer therapeuticagent is selected from T cell agonists, immune checkpoint modulators,STING agonists, RIG-I agonists, other toll-like receptor agonists.

In some embodiments, the additional cancer therapeutic agent is a T cellcostimulatory molecule. In some embodiments, the T cell costimulatorymolecule is selected from OX40, CD2, CD27, CDS, ICAM-1,LFA-1/CD11a/CD18, ICOS/CD278, 4-1BB/CD137, GITR, CD30, CD40, BAFFR,HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, and CD83, or aligand thereof. In some embodiments, a ligand of a costimulatorymolecule is an antibody specifically binding to the costimulatorymolecule. In particular embodiments, the additional cancer therapeuticagent is selected from an anti-OX40 antibody, an anti-OX40L antibody, ananti-ICOS antibody, an anti-CTLA4 antibody, an anti-CD40L antibody, ananti-CD28 antibody, an anti-LFA1 antibody, an anti-TIM1/TIM3 antibody,anti-LAG3 antibody, anti-Siglec-15 antibody, an anti-PD1 antibody, ananti-PDL1 antibody, an anti-CD27 antibody and an anti-4-1BB antibody.

In some embodiments, the additional cancer therapeutic agent is atumor-associated antigen produced by the cancer that is being preventedor treated with the method. In some embodiments, the cancer beingprevented or treated is leukemia, lymphoma, melanoma, colorectal,breast, prostate, renal, pancreatic, head and neck, skin, and braincancer, lung cancer, and the tumor-associated antigen is selected fromCD19, CD20, CD22, CD38, CD138, CD30, CD52, CD56, CD79, CD123, CD206,CD303, CD304, EGFR, folate receptor alpha, folate receptor beta,mesothelin, Her2, transferrin receptor, and PSMA. In some embodiments,the additional cancer therapeutic agent is an immune checkpointmodulator selected from inhibitors of PD-1, PD-L1, PD-L2, TIM-3, LAG-3,CEACAM-1, CEACAM-5, CLTA-4, VISTA, BTLA, TIGIT, LAIR1, CD47, SIRP-α,CD160, 2B4, CD172a, and TGFR. In particular embodiments, the additionalcancer therapeutic agent is a PD-1 inhibitor. In particular embodiments,the additional cancer therapeutic agent is a PD-L1 inhibitor. Inparticular embodiments, the additional cancer therapeutic agent is aCD47 inhibitor. In some embodiments, the additional cancer therapeuticagent is an antibody specifically binding to the immune checkpointmodulator. In particular embodiments, the additional cancer therapeuticagent is an anti-PD-1 antibody or an antigen-binding fragment thereof.In particular embodiments, the additional cancer therapeutic agent is ananti-PD-L1 antibody or an antigen-binding fragment thereof. Inparticular embodiments, the additional cancer therapeutic agent is ananti-CD47 antibody or an antigen-binding fragment thereof. In particularembodiments, the additional cancer therapeutic agent is an anti-CD172aantibody or an antigen-binding fragment thereof, In particularembodiments, the additional cancer therapeutic agent is an anti-OX40antibody or an antigen-binding fragment thereof, In particularembodiments, the additional cancer therapeutic agent is an anti-TIM3antibody or an antigen-binding fragment thereof, In particularembodiments, the additional cancer therapeutic agent is an anti-LAG3antibody or an antigen-binding fragment thereof. Anti-PD-1 andanti-PD-L1 antibodies and their uses are described in, for example,US20180030137, U.S. Pat. No. 9,815,898, US20170313776, US20170313774,US20170267762, WO2017019846, WO2018013017, US20180022809, US20180002423,WO2017220990, WO2017218435, WO2017215590, U.S. Pat. No. 9,828,434, andWO2017196867. Anti-CD47 antibodies and their uses are described in, forexample U.S. Pat. Nos. 9,663,575, 9,803,016, US20170283498,US20170369572, WO2017215585, WO2017196793, and WO2017049251.

In some embodiments, the additional cancer therapeutic agent is a STINGpathway agonist. STING (stimulator of interferon genes, also known asTMEM173, MITA, ERIS, and MPYS) is a transmembrane protein localized tothe ER that undergoes a conformational change in response to directbinding of cyclic dinucleotides (CDNs), resulting in a downstreamsignaling cascade involving TBK1 activation, IRF-3 phosphorylation, andproduction of IFN-P and other cytokines. The STING pathway intumor-resident host antigen presenting cells is involved in theinduction of a spontaneous CD8+ T cell response against tumor-associatedantigens. Activation of this pathway and the subsequent production ofIFN-P also contributes to the anti-tumor effect. In some embodiments,the STING pathway agonist is ADU-S100. Additional STING agonists andtheir uses are described in, for example, US20180028553, US20170319680,US20170298139, US20060040887, US20080286296, US20120041057,US20140205653, WO2014179335, WO 2014179760, US20150056224, WO2016096174, WO 2017011444, WO 2017027645, and WO 2017027646.

In some embodiments, the additional cancer therapeutic agent is a RIG-Ipathway agonist. RIG-I (retinoic acid-inducible gene-I) is a member ofpattern-recognition receptors that initiates a host's innate immunesystem to defend against pathogenic microbes in early phases ofinfection. There are three members of the (RIG-I)-like receptors family:RIG-I, MDA5 (melanoma differentiation factor 5), and LGP2 (laboratory ofgenetics and physiology 2), which are expressed in most cell and tissuetypes. RIG-I functions as a cytoplasmic sensor for the recognition of avariety of RNA viruses and subsequent activation of downstream signalingto drive type I IFN production and antiviral gene expressions. ActivatedRIG-I recruits its downstream adaptor molecule MAVS (also known asIPS-1, CARDIF, and VISA) through CARD-CARD-mediated interactions. Theoligomeric RIG-I CARD assembly and the polymeric formation of MAVS,together serve as a signaling platform for protein complexes thatmediate the bifurcation of signaling into two branches. One branchrecruits tumor necrosis factor receptor-associated factors (TRAF)-2/6and the receptor-interacting protein 1 to subsequently activate the IKKcomplex, resulting in NF-κB activation. The other branch signals throughTRAF3 and activates the TANK/IKKγ/IKKϵ/TBK1 complex, leading to thephosphorylation and dimerization of interferon regulator factors (IRF)-3and -7. Liu et al., Front Immunol. 2017, 7:662. Activation of thispathway contributes to the anti-tumor effect. In some embodiments, theRIG-I pathway agonist is RGT100. RIG-I agonists and their uses aredescribed in, for example, US20170057978, US20170258897, U.S. Pat. Nos.9,381,208, 9,738,680, 9,650,427, WO2017173427, and WO2017011622.

In some embodiments, the additional cancer therapeutic agent is atoll-like receptor agonist selected from TLR1 agonist, TLR2 agonist,TLR3 agonist, TLR4 agonist, TLR5 agonist, TLR6 agonist, TLR7 agonist,TLR8 agonist, and TLR10 agonist.

In further embodiments, in relation to a method of treating cancer, theCpG-containing immunostimulating polynucleotide is administered (eitherin the free-standing form or as a CpG-Ab immunoconjugate) in combinationwith one or more additional therapeutic agents or procedures, forexample wherein the additional therapeutic agent or procedure isselected from the group consisting of chemotherapy, a targetedanti-cancer therapy, an oncolytic drug, a cytotoxic agent, animmune-based therapy, a cytokine, surgical procedure, a radiationprocedure, an activator of a costimulatory molecule, an inhibitor of aninhibitory molecule, a vaccine, a cellular immunotherapy, a cell-basedtherapy (e.g., CAR-T, TILs, TCR-T, CAR-NK, and CAR-macrophage therapies)and an oncolytic virus therapy.

EXAMPLES

The presently disclosed subject matter will be better understood byreference to the following Examples, which are provided as exemplary ofthe invention, and not by way of limitation.

Materials

Prototype peptides were made in house, but can be purchased at custompeptide suppliers (e.g. CPC Scientific). Oligonucleotides were madein-house or by LGC. Transglutaminase used in these examples wereisolated from various bacterial Streptoverticillium strain (e.g.,Ajinomoto). The Q-tag mAbs were produced at Sino Biologicals orinternally.

Production of Oligonucleotides

Oligonucleotides were generally prepared in accordance with the solidphase synthesis scheme shown below, beginning with an initialdeprotection of the solid support for the oligonucleotide synthesis,followed by coupling of the solid support with to the first nucleotide,thiolation to give the phosphothioester and repeated deprotection andcoupling to give the entire oligonucleotide sequence.

The general synthesis of oligonucleotides as provided herein isdescribed below.

Deprotection: A dimethoxytrityl-1,3-propanediol glycolate protectedcontrolled pore glass solid support (DMTO-C3-CPG, 1000A, Bulk Density0.26-0.36 g/cc, Loading 30-40 μmol/g) was reacted with 3% dichloroaceticacid in toluene (v/v) at 25° C., to give the deprotected CPG support. UVabsorption of an aliquot of the reaction mixture was measured toidentify the reaction endpoint (wavelength 350 nm, target minimumabsorbance 0.25 OD, using a fixed watch command setting) and to confirmremoval of the dimethoxytrityl protecting group.

Activation Coupling: The deprotected CPG support was coupled with thefirst nucleotide phosphoramidite precursor for the 3′-end, for therespective oligonucleotide to be synthesized, by adding and mixing thedesired 3′ nucleotide (3 equiv.) for 5 minutes at 25° C. to the reactorcontaining the deprotected CPG support in the presence of an activator5-Ethylthio-1H-tetrazole (0.5M in ACN) at 60% of the nucleotideconcentration.

Thiolation Sulfurization: Following the coupling step, the linkingphosphite triester moiety of the added nucleotide precursor wasthiolated (or sulfurized) by adding Polyorg Sulfa (3-phenyl1,2,4-dithiazoline-5-one), 0.15M in dry ACN, to give thephosphothioester.

Capping: After sulfurization, the CPG support and linked nucleotide weretreated with two capping compositions (Capping composition A: 20%N-methylimidazole in ACN; Capping B composition B: 20% Acetic Anhydride,30% Pyridine, 50% ACN) to block unreacted nucleotide reactants.

Repeat Synthesis: The remaining nucleotides were added in sequence fromthe 3′ end to the 5′ end, employing the appropriate phosphoramiditeprecursors in solution, by repeating the steps of deprotection,activation/coupling, thiolation/sulfurization and capping as describedabove to obtain the desired oligonucleotide sequence in protected form.All phosphoramidite prescursors were mixed with the CPG support for 5minutes during the coupling step, except for dT-Thiophosphoramidite,which was mixed for 15 minutes.

Selected phosphoramidite precursors used in the synthesis are shownbelow. The phosphoramidite precursors were prepared in solutions withthe solvents and at the concentrations, respectively shown, to be usedin the coupling steps.

Amidite Structure Concentration DMT-dC(Ac) Amidite

0.1M in dry ACN: DMT-dG(dmf) Amidite

0.1M in dry ACN: DMT-dT phosphoramidite

0.1M in dry ACN: Fmoc-protected DMT- dT PEG2 NH2 Amidite

0.1M in dry ACN 5-Br-dU-CE Phosphoramidite

0.1M in dry ACN dT- Thiophsophoramidite

0.15M in dry 10% (v/v) DCM/ACN 2′-O-Methyl 5- 0.1M in dry ACN MethylUridine CED phosphoramidite dG-Thiophosphoramidite

0.1M in dry ACN

Exemplary Fmoc-protected oligonucleotide compounds 6.1a, 6.2a and 6.3aobtained from the synthesis steps described above are shown below. Thedeprotection, purification and coupling of compound 6.1a to prepare thecompound 6.1b is further described below.

Fmoc-Protected, CPG-Supported Compound 6.1a

Fmoc-Protected, CPG-Supported Compound 6.2a

Fmoc-Protected, CPG-Supported Compound 6.3a

The Fmoc-protected, CPG-supported oligonucleotide compound 6.1a obtainedfrom the synthesis above was simultaneously cleaved from the support anddeprotected by reacting the CPG support with 20 mM dithiothreitol inammonium hydroxide:methylamine, 1:1 (v/v) for 2 hours at roomtemperature to give crude compound 6.1a. The crude product was purifiedby ion-pair reversed phase HPLC (IP-RP-HIPLC) and its identity confirmedby ESI-MS. Crude compound 6.1a was purified by HPLC and desalted.

Compound 6.1a was subsequently reacted withO-[2-(Fmoc-amino)-ethyl]-O′-[3-(N-succinimidyloxy)-3-oxopropyl]polyethyleneglycol (Fmoc-N-amido-dPEG24-NHIS ester) in sodium bicarbonate buffer togive Fmoc-protected compound 6.1b. Fmoc-protected compound 6.1b wasreacted with 20 mM dithiothreitol in ammonium hydroxide:methylamine, 1:1(v/v) for 2 hours at room temperature to give crude compound 6.1b. Thecrude product was purified by ion-pair reversed phase HIPLC(IP-RP-HIPLC) and its identity confirmed by ESI-MS. Crude compound 6.1bwas purified by HPLC, desalted, and lyophilized to give the purifiedoligonucleotide 6.1b.

Production of Antibodies

Antibodies generated in-house are typically expressed in suspensionculture of Expi293 system (ThermoFisher) according to the manufacturer'smanual. The expressed antibodies are purified via Protein A captureusing MabSelectLX chromatography (GE), elution with 0.1M citrate (pH3.3) and dialyzed in final buffer composition of 1×PBS (PhosphateBuffered Saline, pH 7.4).

One-Step Conjugation Method Via mTG (Microbial Transglutaminase)

Q-tag with the sequence RPQGFGPP (SEQ ID NO: 49) was genetically linkedto the C-terminus of the heavy chain of antibody. To performconjugation, the purified antibody (containing the engineered Q tags atthe C-terminal of heavy chain) were first buffer exchanged into 25 mMTris, 150 mM NaCl pH 8. The Ab-Q-tag containing moiety and CpG wereadded in molar ratio of 1:1.3 and incubated overnight with a finalconcentration of 1% mTG (w/v) (Ajinomoto) at room temperature. Finalconcentration of antibody used for conjugation is generally ˜20-25 uM.Mixture was loaded to a Q Sepharose HP (GE) equilibrated in 20% Buffer B(40 mM Tris, 2M NaCl pH8) and 80% Buffer A (40 mM Tris, pH8). Column waswashed with 5 column volumes of 20% Buffer B. Separation was achievedwith using a linear gradient from 20% B to 60% B in 30 column volumes.DAR1 peak fractions (Q tag conjugated with one CpG moiety) were pooledand concentrated followed by a gel filtration step using S200 (GE).Monomeric peak fractions were pooled and concentrated.

Biological Evaluation of CpG-Nucleotides and Antibody-CpG NucleotideConjugates

Trima residuals were received from Vitalant and diluted 1:4 withPhosphate Buffered Saline (PBS, Gibco). Diluted blood was split into twotubes and underplayed with 15 mL Ficoll-Paque (GE Healthcare). Tubeswere centrifuged for 30 minutes at 400×g. PBMCs were collected from theinterface and resuspended in FACS buffer (PBS with 0.5% Bovine SerumAlbumin (Gibco)). B cells were purified by negative selection using theB Cell Isolation Kit II, human (Miltenyi Biotec) and LS columns(Miltenyi Biotec) according to manufacturer's protocol.

PBMCs were immediately plated onto a 96-well format (500K/well) inComplete RPMI (RPMI+10% FBS). Five-fold serial dilutions were added tothe cells from 100 nM to 6.4 μM of antibody and conjugated antibody and1 uM to 64 μM of CpG polynucleotides at 37° C. under 5% CO2 for 48 to 96hours. Cells were pelleted by centrifugation for five minutes at 400×gand stained at 4° C. in Fixable Viability Dye eFluor 780 (Thermo Fisher)diluted 1:4000 in PBS. Cells were centrifuged and stained at 4° C. inFACS buffer for 30 minutes containing FcR Blocking Reagent (MiltenyiBiotec), anti-CD19, anti-CD20, anti-CD40, anti-HLADR and anti-CD80 for Bcell assays and anti-CD14, anti-CD3, anti-CD19, anti-CD14, anti-CD123,anti-CD11c and anti-CD86 for pDC assays. Cells were centrifuged andwashed twice in FACS buffer and fixed in 0.5% paraformaldehyde.CountBright™ Absolute Counting Beads (Thermo Fisher) were added to eachwell to count the number of cells. Cells were analyzed on Attune NxTFlow Cytometer (Thermo Fisher), with subsequent data analysis by Flowjo10.7 (Treestar). Dead cells were excluded by gating on the eFluor780-negative population. Lineage specific cells were first excluded(CD19, CD3, CD14) prior to gating CD123+CD11c− cells to identify pDC andgating CD19+, CD20+ or CD19+CD20+ cells to identify B cells.

Example 1: Activities of Free Immunomodulating Oligonucleotides (CpGs)in Human PBMCs

Human PBMCs were treated with free CpGs (SEQ ID NOs: 3 and 26-28) toevaluate their respective activities as observed by HLADR and CD40expression on CD19 positive B cells (as shown in FIGS. 1A-1B). CpGs (SEQID NO: 26-28) all showed enhanced activities compared with CpG (SEQ IDNO: 3).

Example 2: Activities of Immunomodulating Polynucleotides and theirRespective Antibody Conjugates

Various CpG polynucleotides, SEQ ID NO: 3-25, were tested for theireffects on proliferation and/or activation of B cells. FIGS. 2A-2C showthe respective activities of select CpGs alone. All CpG polynucleotidestested enhanced the activation of B cells after 48 hours of incubation.As determined by counting beads to calculate absolute B cell number andCD40 expression, all CpGs increased the number of B cells and CD40expression. A select number of CpG polynucleotides tested showedenhanced effects on B-cell proliferation and activation compared withCpG (SEQ ID NO: 3).

As an antibody-CpG conjugate (FIG. 2D) to anti-CD22 antibody (SEQ ID NO:56 and SEQ ID NO: 57), all the respective conjugates of modified CpGs(SEQ ID NOS: 9, 12, 13, 15, and 20) have increased B cell activation,compared with the respective conjugate of CpG (SEQ ID: NO 3) and nakedCpG (SEQ ID NO: 3), as determined by CD80 expression.

Example 3: Transglutaminase-Mediated Conjugation

The transglutaminase-mediated conjugation was tested using anoligonucleotide A (with the sequence: tucgtcgtgacgtt, SEQ ID NO: 1)coordinated to a PEGylated linker (—NH—C(═O)—PEG₂₃-NH₂, structure shownbelow), and Q-tag peptides sequences SEQ ID NOs: 39-47 and 50-52.

2 nmol of the Q-tag was added to 1 nmol of the linker in the present of0.04 nmol of transglutaminase in PBS. The final concentration of linkeris 50 μM. Reactions were kept at room temperature and quenched with 8 Mformamide at 1 hour. The reaction solution was analyzed usingreverse-phase HPLC with Xbridge C18 column (4.6×150 mm) using solvent A(50 mM TEAA in water) and solvent B (Acetonitrile) with a gradient of20% to 60% of solvent B in 10 minutes at 60° C. Alternatively, thereaction solution was analyzed using reverse-phase HPLC with Luna 3μ C18column (4.6×50 mm) using solvent A (0.1% TFA in water) and solvent B(0.1% TFA in Acetonitrile) with a gradient of 10% to 70% of solvent B in10 minutes at 50° C.

FIG. 3 shows the yields of the transglutaminase-mediated conjugation andpeptide deamidation with various Q-tags. RPQGF (SEQ ID NO:47), RPQQF,RPRPQQF showed high conjugate percentage and moderately low deamidation

FIGS. 4A-4B show the conjugation and deconjugation of two conjugatesprepared from Q-tag with SEQ ID NOs: 39 and 47 over time. RPQGF (SEQ IDNO:47) has higher percentage of conjugation with all Q-tag: linker+CpGratio tested, over a duration of 16 hrs. Moreover, the deconjugationrate of RPQGF (SEQ ID NO:47) is also slower compared with LSLSPGLLQGG(SEQ ID NO:39).

Example 4: Activities of Anti-CD22 and Anti-BDCA2 Antibody/CpGConjugates

Q-tag with the sequence RPQGFGPP (SEQ ID NO: 49) was genetically linkedto the C-terminus of the heavy chain of antibody. To performconjugation, the purified antibody (containing the engineered Q tags atthe C-terminal of heavy chain) were first buffer exchanged into 25 mMTris, 150 mM NaCl pH8. The Ab-(Q-tag):CpG containing moiety added inmolar ratio of 1:1.3 were mixed and incubated overnight with a finalconcentration of 1% mTG (w/v) (Ajinomoto) at room temperature. Finalconcentration of antibody used for conjugation is generally ˜20-25 uM.Mixture was loaded to a Q Sepharose HP (GE) equilibrated in 20% Buffer B(40 mM Tris, 2M NaCl pH8) and 80% Buffer A (40 mM Tris, pH8). Column waswashed with 5 column volumes of 20% Buffer B. Separation was achievedwith using a linear gradient from 20% B to 60% B in 30 column volumes.DAR1 peak fractions (Q-tag conjugated with one CpG moiety) were pooledand concentrated followed by a gel filtration step using S200 (GE).Monomeric peak fractions were pooled and concentrated.

Trima residuals were received from Vitalant and diluted 1:4 withPhosphate Buffered Saline (PBS, Gibco). Diluted blood was split into twotubes and underplayed with 15 mL Ficoll-Paque (GE Healthcare). Tubeswere centrifuged for 30 minutes at 400×g. PBMCs were collected from theinterface and resuspended in FACS buffer (PBS with 0.5% Bovine SerumAlbumin (Gibco)).

PBMCs were immediately plated onto a 96-well format (500K/well) inComplete RPMI (RPMI+10% FBS). Five-fold serial dilutions were added tothe cells from 100 nM to 6.4 μM of antibody and conjugated antibody and1 uM to 64 μM of CpG polynucleotides at 37° C. under 5% CO2 for 48 to 96hours. Cells were pelleted by centrifugation for five minutes at 400×gand stained at 4° C. in Fixable Viability Dye eFluor 780 (Thermo Fisher)diluted 1:4000 in PBS. Cells were centrifuged and stained at 4° C. inFACS buffer for 30 minutes containing FcR Blocking Reagent (MiltenyiBiotec), anti-CD19, anti-CD20, anti-CD40, anti-HLADR and anti-CD80 for Bcell assays and anti-CD14, anti-CD3, anti-CD19, anti-CD14, anti-CD123,antiCD11c and anti-CD86 for pDC assays. Cells were centrifuged andwashed twice in FACS buffer and fixed in 0.5% paraformaldehyde.CountBright Absolute Counting Beads (Thermo Fisher) were added to eachwell to count the number of cells. Cells were analyzed on Attune NxTFlow Cytometer (Thermo Fisher), with subsequent data analysis by Flowjo10.7 (Treestar). Dead cells were excluded by gating on the eFluor780-negative population. Lineage specific cells were first excluded(CD19, CD3, CD14) prior to gating CD123⁺CD11c⁻ cells to identify pDC andgating CD19⁺, CD20⁺ or CD19⁺CD20⁺ cells to identify B cells.

Interferon alpha levels were assayed using LegendPlex human inflammationpanel 1 (Biolegend). Supernatant from pDC assays were collected afterpelleting the cells.

Various B-cell and pDC specific antibodies were conjugated with CpG12070 (SEQ ID NO: 3), CpG (SEQ ID NOs: 9, 12, 20, 27 and 28), all withthe DAR1 configuration, at their C-terminus heavy chain Q-tag (RPQGFGPP)(SEQ ID NO: 49) via transglutaminase reaction. The conjugated antibodieswere subsequently tested for their effects on proliferation and/oractivation. The anti-CD22 antibodies conjugated with CpG are RFB4 (SEQID NOs: 56 and 57). As shown in FIGS. 5A-5D, the anti-CD22-CpGantibodies enhanced the activation of B cells as compared to naked CpGpolynucleotide 12070 (SEQ ID NO: 3) and human IgG control antibody after96 hours. As determined by CD19, CD40, CD69, CD86, and HLADR expression,only anti-CD22-CpG and CpG alone induced B cell activation. Directdelivery of CpG by a targeting antibody results in higher activation ascompared to CpG alone. Moreover, anti-CD22 conjugated with CpG (SEQ IDNO: 9 and 20) has enhanced activity compare with anti-CD22 conjugatedwith CpG (SEQ ID NO: 3).

pDC targeting antibody, anti-BDCA2 (SEQ ID NOs: 111 and 112 for VH andVL sequences, respectively), was similarly conjugated with CpG 12070(SEQ ID NO: 3), CpG (SEQ ID NO: 9, 12, 20, 27 and 28), all with the DAR1configuration, and tested for its effects on activation as comparable tounconjugated antibody after 48 hours observed by CD86, CD40 and HLADRexpression (FIGS. 6A-6C) and by CD40 expression in monocytes and mDCs aswell as CD19, CD40, CD3 and CD69 expression (FIGS. 7A-7D). The CpG12070:anti-BDCA2 conjugate also induced IFNα expression in human PBMCs(FIG. 6D).

Conjugated anti-pDC-CpG antibodies and CpG alone induced HLADRexpression as compared to antibody alone.

BDCA VH sequence: (SEQ ID NO: 111)DVQLVESGGGLVKPGGSLRLSCAASGFTFSTYTMSWVRQAPGKGLEWVATISPGDSFGYYYPDSVQGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCTRDIYYNYGAWFAYWGQGTLVTVSS BDCA-VL sequence: (SEQ ID NO: 112)DIQLTQSPSSLSASVGDRVTITCKASQSVDYDGDSYMNWYQQKPGKAPKLLIYAASTLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCAEDRTFGQ GTKVEIK

Example 5: Development of Humanized Anti-CD22 Antibodies

This Example describes the generation of humanized anti-CD22 antibodies.The parental antibody was a mouse anti-CD22 antibody (SEQ ID Nos: 56 &57 for VH and VL sequences, respectively).

Mouse anti-CD22 VH sequence (SEQ ID NO: 56)EVQLVESGGGLVKPGGSLKLSCAASGFAFSIYD MSWVRQTPEKRLEWVAYISSGGGTTYYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARH SGYGSSYGVLFAYWGQGTLVTVSSMouse anti-CD22 VL sequence (SEQ ID NO: 57)DIQMTQTTSSLSASLGDRVTISCRASQDISNYL NWYQQKPDGTVKLLIYYTSILHSGVPSRFSGSGSGTDYSLTISNLEQEDFATYFCQQGNTLPWTFG GGTKLEIK

4 variants of the VH domain (RH1, RH2, RH3, and RH4; SEQ ID Nos: 64-67,respectively) and 5 variants of the VL domain (RL1, RL2, RL3, RL4, andRL5; SEQ ID Nos:68-72, respectively) were designed. The alignments ofthe respective VH and VL domains are shown in FIGS. 8A-8B. Frameworksequences used for each variant were as follows: RH1-human IGHV3;RH2—human IGHV3 with some mutations; RH3—FW1—3 based on IGHV3—48*03 withFW4 as parental mouse; RH4—human IGHV4; RL1—human IGLV1; RL2—human IGLV1with some mutations; RL3—FW1—3 based on IGKV1—39*01 with FW4 as parentalmouse; RL4—IGKV3; RL5—IGVK2 (FW1 and FW2 flank CDR1; FW2 and FW3 flankCDR2, and FW4 is after CDR3). A total of 20 antibody constructs weregenerated by combining each VH domain with each VL domain, and using thehuman IgG1 AAA Fc region with a C-terminal Q-tag (RPQGFGPP) (SEQ ID NO:49) in the antibody heavy chain constant domain (SEQ ID NO:95), as shownbelow. IgG1 AAA Fc contains L234A, L235A, and G237A substitutions, aminoacid position numbering according to EU index.

Construct Name Light chain + Heavy chain TNT69 RL1_hKappa +RH1_hIgG1_AAA_Qtag TNT70 RL1_hKappa + RH2_hIgG1_AAA_Qtag TNT71RL1_hKappa + RH3_hIgG1_AAA_Qtag TNT72 RL1_hKappa + RH4_hIgG1_AAA_QtagTNT73 RL2_hKappa + RH1_hIgG1_AAA_Qtag TNT74 RL2_hKappa +RH2_hIgG1_AAA_Qtag TNT75 RL2_hKappa + RH3_hIgG1_AAA_Qtag TNT76RL2_hKappa + RH4_hIgG1_AAA_Qtag TNT77 RL3_hKappa + RH1_hIgG1_AAA_QtagTNT78 RL3_hKappa + RH2_hIgG1_AAA_Qtag TNT79 RL3_hKappa +RH3_hIgG1_AAA_Qtag TNT80 RL3_hKappa + RH4_hIgG1_AAA_Qtag TNT81RL4_hKappa + RH1_hIgG1_AAA_Qtag TNT82 RL4_hKappa + RH2_hIgG1_AAA_QtagTNT83 RL4_hKappa + RH3_hIgG1_AAA_Qtag TNT84 RL4_hKappa +RH4_hIgG1_AAA_Qtag TNT85 RL5_hKappa + RH1_hIgG1_AAA_Qtag TNT86RL5_hKappa + RH2_hIgG1_AAA_Qtag TNT87 RL5_hKappa + RH3_hIgG1_AAA_QtagTNT88 RL5_hKappa + RH4_hIgG1_AAA_Qtag TNT89 Control_hRFB4_LC_hKappa +Control_hRFB4_HC_hIgG1_AAA_Qtag TNT90 Control_hRFB4 +mutCDRL1_LC_hKappa + Control_hRFB4 + mutCDRH3_HC_hIgG1_AAA_ Qtag TNT31Mouse_RFB4

For antibody selection, the following factors were considered: CD22binding affinity, CD22 biological activity, binding to cynomolgus CD22,expression in CHO or Expi293 cells, conjugation yield, SEC-HIPLC profile(e.g., aggregation, peak shape), VH/VL pairing, sequence liabilities,and stability testing (e.g., freeze-thaw and thermostability).

Each construct was expressed in a 293FS cell line. As shown in FIG. 9 ,antibodies with RL4 and RL5 had the lowest overall expression levels.RH4 also decreased overall expression level in each light chain pairing.

Binding of humanized anti-CD22 antibodies to human CD22 was measured bysurface plasmon resonance (SPR). For assessment of binding by SPR,biotinylated protein A (15 μg/mL) was immobilized to the surface of anNLC chip. 30 μL of mAb supernatant was added to 150 μL of PBS-T andcaptured over the protein A surface. Serial dilutions of human CD22-gly(300 nM, 3× dilution) were injected over the mAb-coated chips, andbinding kinetics were determined. Chips were regenerated using 4:1 v/vof Pierce IgG elution butter/4M NaCl. Binding data are provided below.

Sample ka kd KD Name 1/Ms 1/s M TNT69 2.01E+05 1.26E−04 6.24E−10 TNT702.35E+05 1.03E−04 4.39E−10 TNT72 2.23E+05 1.46E−04 6.55E−10 TNT712.81E+05 1.46E−03 5.20E−09 TNT73 1.58E+05 2.30E−03 1.45E−08 RFB42.97E+05 8.22E−04 2.77E−09 TNT74 1.86E+05 2.89E−03 1.56E−08 TNT761.99E+05 6.01E−03 3.02E−08 TNT75 2.80E+05 8.40E−03 3.00E−08 TNT772.03E+05 2.07E−03 1.02E−08 TNT78 1.95E+05 4.85E−03 2.49E−08 TNT802.23E+05 5.19E−03 2.33E−08 TNT79 2.70E+05 6.29E−03 2.33E−08 TNT811.69E+05 1.17E−04 6.92E−10 TNT82 1.67E+05 1.56E−04 9.37E−10 TNT842.71E+05 6.67E−04 2.46E−09 TNT83 2.69E+05 1.60E−03 5.94E−09 RFB43.06E+05 8.02E−04 2.62E−09 TNT85 1.35E+05 1.69E−04 1.25E−09 TNT861.30E+05 1.41E−04 1.09E−09 TNT88 2.15E+05 2.89E−04 1.34E−09 TNT872.50E+05 1.71E−03 6.85E−09 TNT89 3.59E+05 1.29E−03 3.60E−09 TNT903.55E+05 5.23E−05 1.47E−10

All of the humanized RFB4 variants bound to human CD22 (d1 to d7 domainaccording to NP_001762.2) with affinities between ˜30 nM and ˜0.1 nM.The affinity of the parental RFB4 antibody binding to human CD22 was ˜3nM. TNT71, TNT84, TNT83, TNT85, TNT86, TNT88, TNT89, and TNT90 hadaffinities equivalent to RFB4. TNT69, TNT70, TNT72, TNT81, TNT82, TNT90have higher affinity (˜0.1 nM) than RFB4.

Binding of humanized anti-CD22 antibodies to human or cynomolgus PBMCswas measured by flow cytometry. Trima residuals were received fromVitalant and cynomolgus monkey whole blood were received from BioIVT.Both were diluted 1:4 with Phosphate Buffered Saline (PBS, Gibco).Diluted blood was split into two tubes and underplayed with 15 mLFicoll-Paque (GE Healthcare). Tubes were centrifuged for 30 minutes at400×g. PBMCs were collected from the interface, washed and resuspendedin FACS buffer (PBS with 0.5% Bovine Serum Albumin (Gibco)). 100K PBMCsfrom human and cynomolgus monkey were plated in 96-well plates andpelleted by centrifugation for five minutes at 400×g and stained at 4°C. in 100 μl Fixable Viability Dye eFluor 780 (Thermo Fisher) diluted1:4000 in PBS. Cells were centrifuged and stained at 4° C. in 100 μlFACS buffer for 30 minutes containing FcR Blocking Reagent (MiltenyiBiotec), anti-CD20 and Alexa Fluor 647 conjugated CD22 bindingantibodies, which were labeled using Molecular Probes Alexa Fluor 647Protein Labeling kit (Molecular Probes) according to manufacturer'sprotocol. Cells were centrifuged and washed twice in 200 μl FACS bufferand fixed in 100 μl 0.5% paraformaldehyde. Cells were analyzed on AttuneNxT Flow Cytometer (Thermo Fisher), with subsequent data analysis byFlowjo 10.7 (Treestar). Dead cells were excluded by gating on the eFluor780-negative population. CD20+ cells were gated to identify B cells andmedian fluorescent intensity for CD22 was determined.

Humanized RFB4 antibodies were evaluated for binding to B-cells fromhuman and cynomolgus monkey. Table below shows the median fluorescentintensity of CD22 when bound by RFB4 humanized antibodies on CD20⁺ andCD20⁻ cells in human and cynomolgus monkey. The fold increase in bindingwas determined by calculating the ratio of CD22 median fluorescentintensity on CD20⁺ over CD20⁻ cells. All humanized antibodies bind CD22on human B cells similar to parental RFB4 but with variability oncynomolgus monkey B cells. As determined by CD22 median fluorescentintensity on human CD20⁺ and CD20⁻ cells, the fold increase in bindingfor CD22 for the humanized antibodies range from 19-26-fold as comparedto 22-fold for parent RFB4. The fold increase in CD22 median fluorescentintensity on cynomolgus monkey CD20⁺ and CD20⁻ for the humanizedantibodies range from 1-16-fold as compared to

Human Cyno CD20⁺ CD20⁻ CD20⁺ CD20⁻ CD22 CD22 CD22 CD22 Median MedianCD22: Median Median CD22: Sample Fluorescent Fluorescent CD20⁺/Fluorescent Fluorescent CD20⁺/ ID Intensity Intensity CD20⁻ IntensityIntensity CD20⁻ TNT 69 1610 63.4 25.4 1412 94.9 14.9 TNT 70 1555 65.923.6 1291 115 11.2 TNT 72 1555 62.1 25.0 1368 109 12.6 TNT 71 1457 65.922.1 1420 101 14.1 TNT 73 1413 70.9 19.9 216 114 1.9 TNT 74 1313 67.119.6 185 105 1.8 TNT 76 1193 58.4 20.4 140 85.4 1.6 TNT 75 1628 60.926.7 588 88.4 6.7 TNT 77 1324 63.4 20.9 236 90.1 2.6 TNT 78 1206 60.919.8 112 90.4 1.2 TNT 80 1211 59.6 20.3 159 86 1.8 TNT 79 1301 60.9 21.4349 90.1 3.9 TNT 82 1515 63.4 23.9 1174 93.2 12.6 TNT 83 1489 64.6 23.01401 130 10.8 TNT 85 1579 68.4 23.1 1375 143 9.6 TNT 86 1572 65.9 23.91428 131 10.9 TNT 88 1531 58.4 26.2 1274 86.7 14.7 TNT 87 1444 60.9 23.71368 85.4 16.0 RFB4 1353 60.9 22.2 960 115 8.3

To assess if CpG conjugation affects the binding of respective humanizedanti-CD22 to human CD22, SPR assay was carried out. The humanizedanti-CD22 antibodies (TNT70, TNT71, TNT72 and TNT74) were conjugatedwith CpG (SEQ ID NO: 3) with a DAR1 configuration and compared withtheir respective naked antibodies. Briefly, biotinylated protein A (15μg/mL) was immobilized to the surface of an NLC chip. 30 nM of purifiednaked mAb or CpG conjugated mAb solution in PBS-T was used for captureover the protein A surface. Serial dilutions of human CD22 (300 nM, 3×dilution) were injected over the mAb-coated chips, and binding kineticswere determined. Chips were regenerated using 4:1 v/v of Pierce IgGelution butter/4M NaCl. Binding data are provided below.

ka kd KD Sample Name 1/Ms 1/s M TNT70 DAR1 1.25E+05 6.29E−05 5.02E−10TNT71 DAR1 8.17E+04 3.14E−05 3.84E−10 TNT72 DAR1 2.01E+05 6.59E−053.28E−10 TNT74 DAR1 1.14E+05 2.10E−03 1.85E−08 TNT71 8.84E+04 2.33E−052.63E−10 TNT72 3.51E+05 3.23E−09 9.22E−10 TNT70 1.40E+05 5.62E−054.00E−10 TNT74 1.10E+05 1.99E−03 1.82E−08

The CpG-conjugated mAbs (TNT70, TNT71, TNT72, and TNT74) bound to humanCD22 with similar affinities to the corresponding naked mAb, indicatingthat CpG conjugation did not alter binding kinetics.

Next, humanized anti-CD22 antibodies were conjugated to CpG 12070, andactivity of antibody conjugates (DAR1) was analyzed. Antibody conjugateswere purified by chromatography under the following conditions: MonoQ5/50 GL; buffer A: 50 mM Tris, pH8; buffer B: 50 mM Tris, 2M NaCl, pH8;2 mL/min, 0-100% B in 200 CV, 1 mL fractions. The results are shown inFIGS. 10A-10D for conjugates to TNT70, TNT71, TNT72, and TNT74,respectively. For purification, only the DAR1 peak was pooled,buffer-exchanged into PBS, and concentrated. Starting material andconjugates were also analyzed in either reduced or non-reduced form bySDS-PAGE (FIG. 10E).

Binding analysis by SPR showed that naked antibodies and CpG conjugatesbound to CD22 with similar affinity.

As shown in FIG. 11 , conjugates to various humanized anti-CD22antibodies were all able to activate B cells. No relationship betweenaffinity to CD22 and B cell activation was observed.

CpG:antibody conjugates were also profiled by size exclusionchromatography (SEC), as shown in FIGS. 12A-12D. Antibody conjugatesincorporating the RH1 VH domain showed broad or double SEC peaks (FIG.12A). Antibody conjugates incorporating the RH2 VH domain werewell-behaved based on SEC analysis (FIG. 12B). Antibody conjugatesincorporating the RH3 VH domain were prone to aggregation, based on SECanalysis (FIG. 12C). Antibody conjugates incorporating the RH4 VH domainwere well-behaved based on SEC analysis but characterized by low overallexpression (FIG. 12D).

Pairings of specific VH and VL domains were analyzed. It was known that,generally, IGHV3 pairs frequently with IGKV1 and IGKV3. Preferredpairings included TNT69, TNT70, TNT71, TNT73, TNT74, TNT75, TNT77,TNT78, TNT79, TNT81, TNT82, and TNT83. Potential sequence liabilities,including residues potentially subject to methionine oxidation,deamidation, and isomerization, were also examined.

Selected characteristics of conjugates tested are summarized in FIG.13A. Antibodies with RH1 behaved abnormally by SEC-HPLC. Antibodies withRH4, RL4, and RL5 had lower expression levels (FIG. 13B). In particular,antibodies with RL4 (IGKV3) had lower expression in CHO cells, as inExpi293 cells. Antibodies with RH3 had a general tendency towards highaggregation. Pairing RH4 and RL5 was generally non-preferred. Based onall characteristics, including expression level and potential sequenceliabilities, pairing the RH2 VH domain with RL1, RL2, or RL3 VL domainwas preferred. TNT70, TNT74, TNT78, and TNT82 were selected for furtherstudy.

To evaluate melting temperature, differential scanning calorimetry (DSC)was used. DSC was performed using a MicroCal VP Capillary DSC (Malvern).DSC is thought to measure heat capacity as a function of temperature.Melting point (Tm) is thought to be a good indicator of thermalstability and a predictor of long-term stability, with more stableproteins having a higher Tm. Results of DSC testing of selectedCpG:antibody conjugates are shown below.

Sample T_(onset) T_(m)1 T_(m)2 TNT70 61.41 71.71 79.26 TNT70 DAR1 64.2371.71 78.09 TNT72 62.64 71.17 86.37 TNT72 DAR1 62.54 70.80 86.00

TNT72 had a higher Tm2 than TNT70. This transition is thought tocorrespond to Fab/CH₃ unfolding. Minor differences in Tm were observedbetween naked and conjugated antibodies. DSC plots are shown in FIG. 14.

Example 6: Analysis of Humanized Anti-CD22 Variants Mutated to RemovePotential Sequence Liabilities

Humanized anti-CD22 antibodies described in Example 5 were next analyzedfor potential sequence liabilities. A potential Asn deamination site ispresent in the CDR-L3 (N92) of all humanized variants (FIG. 8B). Thissite was mutated in the RL1 VH domain to create antibody variants, whichwere tested for the impact of mutation on binding affinity to CD22. Forengineering variants, N92 was mutated to all possible amino acids. Thedata for the mutation to A, C, D, E, F, G, H, L, P, S, T, V, or W isshown below.

Expression of each variant was compared to TNT70 in reducing (FIG. 15B)or non-reducing (FIG. 15A) conditions. Binding properties of the mutantsare summarized below and in FIGS. 15C-15F. All of the N92 substitutionslooked to be tolerable with respect to binding affinity to CD22. Inparticular, N92A, N92D, N92E, and N92F looked to be comparable to theparental antibody, but the KD of all variants looked to be within errorof the assay.

ka kd KD Sample 1/Ms 1/s M TNT70 1.55E+05 3.72E−05 2.40E−10 TNT701.45E+05 7.30E−05 5.04E−10 N92A 1.51E+05 4.45E−05 2.95E−10 N92C 1.11E+057.13E−05 6.43E−10 N92D 1.46E+05 5.05E−05 3.46E−10 N92E 1.47E+05 4.29E−052.93E−10 N92F 1.55E+05 4.58E−05 2.95E−10 N92G 1.51E+05 6.80E−05 4.52E−10N92H 1.49E+05 6.08E−05 4.09E−10 N92K 1.45E+05 6.25E−05 4.31E−10 N92P1.12E+05 7.24E−05 6.44E−10 N92S 1.52E+05 7.14E−05 4.68E−10 N92T 1.49E+057.85E−05 5.29E−10 N92V 1.45E+05 8.32E−05 5.73E−10 N92W 1.43E+05 7.87E−055.49E−10

Example 7: Pharmacokinetics of CpG-Antibody Conjugates

Method: Balb/c mice were obtained from Charles River and used for singledose PK study. Each compound conjugate to RFB4 antibody (SEQ ID NO: 56and SEQ ID NO: 57 for VH and VL domain sequences, respectively) wasformulated at a working dose of 2 mg/mL and each mouse was given 200 mgexcept for Cmpd 4.2b (SEQ ID NO:12) conjugated to RFB4, which was at 170mg. The RFB4 conjugates were administered intravenously via the mousetail vein. Three mice were dosed for each RFB4 conjugate. After dosing,mice had blood withdrawn at the following seven time points: 0.5, 6, 24,48, 72, 144, and 192 hours. Approximately 100 μl of whole blood wascollected into microtainer tubes by orbital bleed. Whole blood sampleswere rested for 30 minutes to allow serum separation. Samples were thencentrifuged for 10 minutes at 4 degree at 10000×g. Serum was transferredto a 1.5 ml tube and frozen until analysis. Immulon 96 well ELISA plates(Thermo Fisher Scientific, cat. #3855) were coated overnight with 1ug/ml, 100 ul/well, sheep anti-human IgG antibody (The Binding Site,cat. #AU003.M) in PBS for anti-human IgG antibody capture or with 2ug/ml, 100 ul/well, NeutrAvidin Biotin Binding Protein (Thermo FisherScientific, cat. #31050) in PBS for anti-BrdU capture. Plates werewashed with Tris-Buffered Saline Tween-20 (TBST, 25 mM Tris, 0.15 MNaCl, 0.05% Tween-20, pH 7.5) and blocked for 1 hour with assay buffer(PBS, 1% BSA, 0.05% Tween-20, 0.25% CHAPS, 5 mM EDTA, 0.35 M NaCl) foranti-human IgG antibody capture and with casein blocker (Thermo FisherScientific, cat. #37528) for anti-BrdU capture. Plates were washed withTBST. 1 ug/ml, 100 ul/well, of biotinylated anti-BrdU mouse monoclonalantibody (BioLegend, cat. #317904) in assay buffer (PBS, 1% BSA, 0.05%Tween-20, 0.25% CHAPS, 5 mM EDTA, 0.35 M NaCl)+20% casein blocker wasadded to plates and incubated for 1 hour at room temperature with gentleshaking at 420 rpm on a plate shaker. Plates were washed with TBST.Serum samples were diluted at a minimum of 1:50 in assay buffer+20%casein blocker (Thermo Scientific, cat. #37528) or RFB4 standard curveprotein with and without various CpGs attached (two-fold serialdilutions from 100 to 0.2 ng/ml, in 1:50 normal mouse serum diluted inassay buffer+20% casein blocker) were added to blocked plates for 1 hourat room temperature with gentle shaking at 420 rpm on a plate shaker.Plates were washed with TBST. Standard curves and samples were incubatedwith 0.2 ug/ml goat anti-human IgG-HRP (Bethyl, cat. #A80-319P) foranti-human IgG antibody capture or 0.4 ug/ml goat anti-human IgG-RP foranti-BrdU, for 1 hour at room temperature with gentle shaking at 420 rpmon a plate shaker. Plates were washed with TBST. All plates wereincubated with 1-Step Ultra TMB ELISA solution (Thermo FisherScientific, cat. #34028) and the reaction was stopped with 0.16 Msulfuric acid solution (Thermo Fisher Scientific, cat. #N600). Plateswere read at an O.D. of 450 nm with a background reference reading at570 nm on a SpectraMax i3 plate reader (Molecular Devices). Proteinconcentrations of serum samples were interpolated from the RFB4 antibodystandard curves with and without various CpGs attached using a 4parameter fit curve and Prism software (GraphPad).

As shown in FIG. 17A, RFB4 conjugated with Cmpd 1.1b (SEQ ID NO:3), Cmpd3.2b (SEQ ID NO:9), Cmpd 4.2b (SEQ ID NO:12), Cmpd 4.3b (SEQ ID NO:13),Cmpd 5.2a (SEQ ID NO:15) or Cmpd 5.7a (SEQ ID NO:20) have similarhalf-life compared to naked RFB4 when total antibody was measured. Thisshows that the conjugation of RFB4 with the respective CpGoligonucleotides does not affect the PK of the antibody. FIG. 17B showsthe PK of RFB4 conjugates by capturing the compounds with anti-BrdUantibody. Surprisingly, when the half-life of RFB4 conjugates isevaluated by capturing the 5′ region of the CpG using anti-BrdUantibody, Cmpd 3.2b (SEQ ID NO: 9), Cmpd 4.2b (SEQ ID NO:12), Cmpd 4.3b(SEQ ID NO:13, Cmpd 5.2a (SEQ ID NO:15) and Cmpd 5.7a (SEQ ID NO:20)have increased half-life compared to Cmpd 1.1b (SEQ ID NO:3).

Example 8: B Cell Activation in Cynomolgus Monkey PBMCs by CpGOligonucleotides

Peripheral blood mononuclear cells (PBMCs) were isolated from cynomolgusmonkey whole blood by Ficoll (GE) separation. 300,000 PBMC cells wereplated in 96 well round bottom plates in RPMI 10% FBS (LifeTechnologies). Cells were treated with 12070 (compound 1.1b, SEQ IDNO:3), CpG 7-7 (Compound 7.7b; SEQ ID NO:35), or media only at astarting concentration of 1 uM followed by 1:5 titration down. Cellswere incubated for 72 hrs prior to staining for flow cytometry. Cellswere centrifuged at 400G for 5 min before media removal. Cells were thenincubated with fixable live dead dye eFluor780 (eBioscience) for 30 minat 4 C. FACS buffer (PBS+2% FBS) was added to the wells beforecentrifugation at 400G for 5 min. Cells were then incubated in human FcRblocking reagent (Miltenyi Biotec) and stained with fluorochrome-labeledantibodies against CD3 (clone SP34, Biolegend), CD14 (clone M5E2,Biolegend), CD16 (clone 3G8, Biolegend), HLADR (clone G46-6, Biolegend),CD1c (clone L161, Biolegend), CD20 (clone 2H7, Biolegend), CD69 (cloneFN50, eBioscience), CD8 (clone RPAT8, Biolegend), CD40 (clone 5C3,Biolegend) and CD86 (clone IT2.2 Biolegend) for 1 hr at 4C. Cells werethen washed twice in FACS buffer before fixing in 0.5% paraformaldehyde.Cells were acquired on an Attune flow cytometer (Thermofisher) withsubsequent analysis using FlowJo Software and tabulated using PrismSoftware.

Cynomolgus monkey PBMCs were stimulated for 72 hrs in presence of 7-7CpG, 12070 CpG or media only at a starting concentration of 1 uMfollowed by 1:5 titration down. Cells were then stained and analyzed byflow cytometry. Following singlet and dead cell exclusion, CD20+ B cellswere gated, followed by median fluorescent intensity of CD86 marker.Treatment with 7-7 induced superior CD86 upregulation (FIG. 18 ),demonstrating enhanced B cell activation compared to 12070.

Example 9: Induction of IL-6 Expression Using Anti-CD22:CpGOligonucleotide Conjugates

Human peripheral blood mononuclear cells (PBMCs) were isolated fromTrima residuals (Vitalant) and diluted 1:4 with Phosphate BufferedSaline (PBS, Gibco). Diluted blood was split into tubes and underlayedwith 15 mL Ficoll-Paque (GE Healthcare). Tubes were centrifuged for 30minutes at 400×g. PBMCs were collected from the interface andresuspended in Complete RPMI (RPMI+10% FBS). One million PBMCs wereplated in 96 round well plates in Complete RPMI and treated withunconjugated anti-CD22 antibody, or anti-CD22 conjugated to 12070 CpG(compound 1.1b, SEQ ID NO:3) starting at a concentration of 100 nMfollowed by 1:5 titration down. Cells were incubated for 48 hrs followedby spin at 400×g for 5 min upon which supernatants were collected. IL6in harvested supernatant were assessed neat using a bead-basedimmunoassay kit (LegendPlex, Biolegend) per manufacturer'srecommendations. Samples were acquired on an Attune flow cytometer(Thermofisher) with subsequent analysis using FlowJo Software andtabulated using Prism Software.

Human PBMCs were stimulated for 48 hrs in the presence of unconjugatedanti-CD22 antibody or anti-CD22 conjugated to 12070 CpG following whichsupernatants were harvested and IL6 was assessed by bead-basedimmunoassay. Anti-CD22 conjugated to 12070 CpG induced robust IL6expression as compared to that after treatment with unconjugatedanti-CD22 antibody (FIG. 19 ), suggesting that anti-CD22 conjugated to12070 can robustly induce IL6 expression from the targeted cell type.

Example 10: Activation of Non-Targeted T and B Cells UsingAnti-SIRP-α:CpG Oligonucleotide Conjugates

Human peripheral blood mononuclear cells (PBMCs) were isolated fromTrima residuals (Vitalant) and diluted 1:4 with Phosphate BufferedSaline (PBS, Gibco). Diluted blood was split into tubes and underlayedwith 15 mL Ficoll-Paque (GE Healthcare). Tubes were centrifuged for 30minutes at 400×g. PBMCs were collected from the interface andresuspended in Complete RPMI (RPMI+10% FBS). One million PBMCs wereplated in 96 round well plates in Complete RPMI and treated withanti-SIRP-α antibody, or anti-SIRP-α conjugated to 12070 CpG (compound1.1b, SEQ ID NO:3) starting at a concentration of 100 nM followed by 1:5titration down. VH and VL domain sequences of the anti-SIRP-α antibodyare provided below.

Anti-SIRP-α VH domain: (SEQ ID NO: 122)EVQLVESGGGVVQPGGSLRLSCAASGFTFSSNAMSWVRQAPGKGLEWVAGISAGGSDTYYPASVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARET WNHLFDYWGQGTLVTVSSAnti-SIRP-α VL domain: (SEQ ID NO: 123)SYELTQPPSVSVSPGQTARITCSGGSYSSYYYAWYQQKPGQAPVTLIYSDDKRPSNIPERFSGSSSGTTVTLTISGVQAEDEADYYCGGYDQSSYTNPFG GGTKLTVL

Cells were then incubated for 48 hrs prior to flow staining. Cells werecentrifuged at 400×g for 5 min before media removal. Cells were thenincubated with fixable live dead dye eFluor780 (eBioscience) for 30 minat 4 C. FACS buffer (PBS+2% FBS) was added to the wells beforecentrifugation at 400×g for 5 min. Cells were then incubated in humanFcR blocking reagent (Miltenyi Biotec) and stained withflurochrome-labeled antibodies against CD3, CD14, CD16, HLADR, CD11c,CD69, CD40, and CD86 for 1 hr at 4 C. Cells were then washed twice inFACS buffer before fixing in 0.5% paraformaldehyde. Cells were acquiredon an Attune flow cytometer (Thermofisher) with subsequent analysisusing FlowJo Software and tabulated using Prism Software.

Human PBMCs were stimulated for 48 hrs in presence of unconjugatedanti-SIRP-α or anti-SIRP-α conjugated to 12070 starting at aconcentration of 100 nM followed by 1:5 titration down. Cells were thenstained and analyzed by flow cytometry. Following singlet and dead cellexclusion, CD20+ B cells (FIG. 20B) or CD3+ T cells (FIG. 20A) weregated, followed by median fluorescent intensity of CD69, an activationmarker. Anti-SIRP-α conjugated to 12070 CpG induced robust activation ofnon-targeted cells such as T cells and B cells, as compared to thatafter treatment with unconjugated anti-SIRP-α, as evident with theupregulation of CD69 (FIGS. 20A & 20B).

Example 11: Treatment with Antibody:CpG Oligonucleotide ConjugatesElicits Robust Immune Memory Response to Tumors

MC38 cells were injected into the right flank of C57BL/6 female mice, ata concentration of 2×10⁶ cells per mouse in DMEM. Tumors were monitoreduntil the average size of tumors reached 150-155 mm³. Mice wererandomized into PBS control, anti-SIRP-α-4523 CpG or anti-mCD22-4523 CpGat 5 mice per cohort. The sequence of the 4523 murine CpGoligonucleotide is tucgtcgtgacgtt-c3, where lower case indicatesphosphothioate linkages, bold indicates iodo-uridine, and underliningindicates phosphotriester linker (SEQ ID NO:121). VH and VL domainsequences of the anti-mCD22 antibody are provided below.

Anti-mCD22 VH domain: (SEQ ID NO: 124)QVQLQQPGAEIVRPGTSVKLSCKASGYTFTDYWMNWVKQRPGQGLEWFGAIDPSDSYTRYNQEFKGKATLTVDTSSTTAYMQLSSLTSEDSAVYFCARSD YTYSFYFDYWGLGTTLTVSSAnti-mCD22 VL domain: (SEQ ID NO: 125)DIVMTQAAFSNPVTLGTSASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCAQNLELP WTFGGGTKLEIK

Anti-SIRP-α-4523 and anti-mCD22-4523 were dosed at 10 mg/kg two times intotal, three days apart. Both drugs were administered intraperitoneally.On day 88, mice with eradicated tumors were re-challenged with MC38(left flank) at 2×10⁶ cells per mouse in DMEM. Naïve mice that have notbeen implanted with MC38 mice were included as control for tumor growth.Tumors were measured in two dimensions with calipers, and tumor volumewas calculated as: length×width×width×0.5, where length was the largerof the two measurements.

On day 88, there were 5 mice with eradicated tumors for each of thegroups treated with either 10 mg/kg anti-SIRP-α 4523 CpG, oranti-mCD22-4523 CpG. Following re-challenge, previously eradicated miceshowed efficient tumor rejection as early as three days post-tumorre-implantation suggesting that treatment with both anti-SIRP-α andanti-mCD22 conjugated to 4523 CpG elicit robust immune memory responseagainst the implanted tumor, not seen in naïve mice who are encounteringMC38 tumor for the first time (FIGS. 21A-21C).

Example 12: Treatment with Anti-Her2:CpG Oligonucleotide ConjugatesLeads to Durable Tumor Eradication

The trastuzumab epitope was integrated into the mouse Her2 gene togenerate mouse/human (m/h) Her2. m/h Her2 expressing MC38 cells wasgenerated by lentiviral transduction and sorted to obtain cells thatexpress m/h Her2. m/h Her2-MC38 cells were injected into the right flankof C57BL/6 female mice, at a concentration of 2×10⁶ cells per mouse inDMEM. Tumors were monitored until the average size of tumors reached 70mm³. Mice were randomized into PBS control, TNT149a (anti-Her2 mIgG2a),and TNT150a (anti-Her2 mIgG1) treatment groups with 5 mice per cohort.Anti-Her2-CpG nucleotide conjugate-treated mice were dosed with 1, 3 and10 mg/kg three times in total, three days apart. Both drugs wereadministered intraperitoneally. Heavy and light chain sequences for theanti-Her2 antibodies are provided below.

Anti-Her2 mIgG2a heavy chain: (SEQ ID NO: 126)EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARTYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGL LQGGAnti-Her 2mIgG1 heavy chain:  (SEQ ID NO: 127)EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARTYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFIYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGLLQGG Anti-Her2 light chain:(SEQ ID NO: 128) DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLILTKDEYERHNSYTCEATHKT STSPIVKSFNRNEC

m/h Her2 MC38-tumor bearing mice were measured and randomized by tumorvolume. On day 4, each cohort of 5 mice had an average tumor size of 70mm³. Mice were treated PBS or 1, 3, and 10 mg/kg of TNT149a or TNT150a.By day 60, 1, 3 and 10 mg/kg TNT149a treated mice dosed three times,three days apart showed tumor eradication (1/5, 5/5 and 5/5 mice,respectively; FIGS. 22A & 22C) while mice treated with 1, 3 and 10 mg/kgTNT150a showed lower number of mice with eradicated tumors (0/5, 3/5 and5/5, respectively; FIGS. 22B & 22D). Mice treated with PBS controlreached endpoint by day 24 and all groups treated with TNT149a orTNT150a showed delayed tumor growth as compared to PBS control. Thesedata show durability in tumor eradication with both anti-Her2-CpG mIgG1and mIgG2a and superior activity in mice treated with anti-Her2-CpGnucleotide conjugate containing Fc effector function.

On day 81, there were 5 mice with eradicated tumors for groups treatedwith 10 mg/kg TNT150a, 3 and 10 mg/kg TNT149a and 3 mice with eradicatedtumors for group treated with 3 mg/kg TNT150a. On day 81, mice witheradicated tumors were rechallenged with m/h Her2 MC38 (lower rightflank), parent MC38 (lower left flank), m/h Her2 B16F10 (upper rightflank) and parent B16F10 (upper left flank) at 2×10⁶ cells (m/h Her2MC38 and MC38 cells) and 1×10⁶ cells (m/h Her2 B16F10 and B16F10 cells)per mouse in DMEM (FIG. 23A). Naïve mice that have not been implantedwith m/h Her2 MC38 mice were included. Tumors were measured in twodimensions with calipers, and tumor volume was calculated as:length×width×width×0.5, where length was the larger of the twomeasurements.

As shown in FIGS. 23B-23I, naïve mice showed growth for all implantedcells. m/h Her2 B16F10, MC38 and m/h Her2 MC38 showed eradicated tumorsor significant delayed tumor growth as compared to naïve. In alltreatment groups, the parent B16F10 tumors grew except in one case, oneof the mouse from the 10 mg/kg Her2 mIgG2a group shows no growth. By day99, all mice showed complete eradication with m/h Her2 MC38 cells. BothMC38 parent and m/h Her2 B16F10 cells showed tumor eradication with theexception of one mouse for both 3 and 10 mg/kg mice previously treatedwith TNT149a for MC38 parent cells and 1-3 mice for all previously 3 and10 mg/kg treated groups. These data show that m/h Her2 MC38 tumorbearing mice with eradicated tumors after treatment with anti-Her2 mIgG1and mIgG2a have potent and durable anti-tumor response to m/h Her2 MC38,parent MC38 and m/h B16F10 but not parent B16F10 tumors. These resultsdemonstrate epitope spreading to eradicate MC38 parental andHer2-expressing B16F10 cells, with slightly better durability seen athigher doses.

Example 13: Treatment with Anti-mCD22:CpG Oligonucleotide ConjugatesLeads to Increased Gene Expression Signatures Related to InterferonSignaling, Antigen Presentation, and Cytotoxicity

CT26 cells were injected into the right flank of BALB/C female mice, ata concentration of 2×10⁶ cells per mouse in RPMI. Tumors were monitoreduntil the average size of tumors reached 270-295 mm³. Mice wererandomized into PBS control, anti-mCD22 and anti-mCD22-CpG with 4 miceper cohort. Anti-mCD22 and anti-mCD22_CpG were administeredintraperitoneally at 10 mg/kg. Tumors were harvested 8 hours posttreatment and processed for RNA. All samples were processed andquantified using the Mouse PanCaner IO 360 Panel (NanoStringTechnologies) per manufacture's protocol by Canopy Biosciences (St.Louis, Mo.). Normalized expression data were analyzed with nSolver andnCounter Advanced Analysis Software and presented as signature scores.The CpG conjugated to anti-mCD22 is 4523 murine CpG oligonucleotide withthe sequence of tucgtcgtgacgtt-c3, where lower case indicatesphosphothioate linkages, bold indicates iodo-uridine, and underliningindicates phosphotriester linker (SEQ ID NO:121).

Normalized expression data of the tumors were analyzed with nSolver andnCounter Advanced Analysis Software to obtain gene expression signaturesas defined by NanoString. CT26-bearing mice treated with anti-mCD22 CpGshowed higher signature scores for interferon signaling (FIG. 24A),antigen presentation (FIG. 24B), and cytotoxicity (FIG. 24C) in thetumor as compared to PBS control and anti-mCD22 alone.

Example 14: Co-Culturing of Murine Bone Marrow-Derived Macrophages andHer2-Positive Tumor Cells in the Presence of Anti-Her2:CpG Conjugateswith Active or Inactive Fc Regions

CSFE labeled targets cells either Her2^(pos) (MC38) or Her2^(neg)(C1498) tumors were co-cultured with mouse-derived macrophages at a 1:2ratio (target:effector) in the presence of unconjugated Her2, TNT149a,TNT150a or 4523 CpG starting at 100 nM diluted 1:2 (6 pts). Cells wereincubated for 24 hrs, spun at 400×g to remove supernatant. Cells werethen washed with PBS and transferred PBS with cells to new plate.Remaining cells in original plate were incubated with TrypLE for 10 min,cells were then scraped and transferred to same plate with PBS andcells. Collected cells were then spun and stained with fixable live deaddye, followed by fluorochrome labeled antibodies to mouse MHCII andCD11b. Samples were acquired on an Attune flow cytometer (Thermofisher)with subsequent analysis using FlowJo Software and tabulated using PrismSoftware.

Bone marrow-derived mouse macrophages were co-cultured in the presenceof Her2^(pos) (MC38) or Her2^(neg) (C1498) tumors and treated withanti-Her2, TNT149a, TNT150a or 4523 CpG for 24 hrs. Median MHCII wasassessed by flow cytometry. Upregulation of MHCII activation marker wasobserved on murine macrophages when co-cultured with Her2^(pos) tumorsin the presence of active Her2-CpG conjugate (TNT149a), but notHer2^(neg) tumors, suggesting that presence of Her2 is required formacrophage activation as evidenced by MHCII induction (FIG. 25 ).Additionally, active Fc (TNT149a) appears to be required for murinemacrophage activation in vitro as evidenced by lack of MHCII inductionwith less active Fc (TNT150a).

Example 15: Induction of B Cell Activation in Human PBMCs byAnti-CD22:CpG Oligonucleotide Conjugates

Human peripheral blood mononuclear cells (PBMCs) were isolated fromTrima residuals (Vitalant) and diluted 1:4 with Phosphate BufferedSaline (PBS, Gibco). Diluted blood was split into tubes and underlayedwith 15 mL Ficoll-Paque (GE Healthcare). Tubes were centrifuged for 30minutes at 400×g. PBMCs were collected from the interface andresuspended in Complete RPMI (RPMI+10% FBS). One million PBMCs wereplated in 96 round well plates in Complete RPMI and treated withanti-CD22 antibody with RH2 VH domain and RL1 N92S VL domain (SEQ IDNos:65 and 87, respectively) conjugated to 7-7 CpG, TNT52 (RFB4anti-CD22 conjugated to 12070), 7-7, 12070 or media only starting at aconcentration of 100 nM for conjugated antibodies or 1 uM for free CpGfollowed by 1:5 titration down. Cells were then incubated for 48 hrsprior to flow staining. Cells were centrifuged at 400×g for 5 min beforemedia removal. Cells were then incubated with fixable live dead dyeeFluor780 (eBioscience) for 30 min at 4C. FACS buffer (PBS+2% FBS) wasadded to the wells before centrifugation at 400×g for 5 min. Cells werethen incubated in human FcR blocking reagent (Miltenyi Biotec) andstained with fluorochrome-labeled antibodies against CD19, CD40, CD80and CD86 for 1 hr at 4C. Cells were then washed twice in FACS bufferbefore fixing in 0.5% paraformaldehyde. Cells were acquired on an AttuneNxT cytometer (Thermofisher) with subsequent analysis using FlowJoSoftware and tabulated using Prism Software.

Human PBMCs were stimulated for 48 hrs in presence of anti-CD22 antibodywith RH2 VH domain and RL1 N92S VL domain (SEQ ID Nos:65 and 87,respectively) conjugated to compound 7.7b (7-7) CpG, TNT52a (RFB4conjugated to 12070), compound 7.7b (7-7) CpG, 12070 CpG, or media only.Cells were then stained and analyzed by flow cytometry. Followingsinglet and dead cell exclusion, CD20+ B cells were gated, followed bymedian fluorescent intensity of CD40 (FIG. 26A), CD80 (FIG. 26B) andCD86 (FIG. 26C) activation markers. Anti-CD22 antibody with RH2 VHdomain and RL1 N92S VL domain conjugated to compound 7.7b (7-7 CpG)displayed higher induction of all activation markers on gated B cellsthat is superior to RFB4-conjugated to 12070. Additionally, free 7.7bCpG induced superior activation of all interrogated markers compared to12070 CpG.

Example 16: Evaluation of Free CpG Activity on CD40 Expression by CD19+B Cells

Materials and Methods

Trima residuals were received from Vitalant and diluted 1:2 withPhosphate Buffered Saline (PBS, Gibco). Diluted blood was split into twotubes and underplayed with 15 mL Ficoll-Paque (GE Healthcare). Tubeswere centrifuged for 30 minutes at 400×g. PBMCs were collected from theinterface, resuspended and washed in FACS buffer (PBS with 0.5% BovineSerum Albumin (Gibco)). After one wash, PBMCs were resuspended inComplete RPMI (RPMI+10% FBS).

PBMCs were immediately plated onto a 96-well format (500K/well) inComplete RPMI. Five-fold serial dilutions were added to the cells from 1uM to 64 μM of CpG polynucleotides at 37° C. under 5% CO2 for 48 hours.Cells were pelleted by centrifugation for five minutes at 400×g andstained at 4° C. in Fixable Viability Dye eFluor 780 (Thermo Fisher)diluted 1:4000 in PBS. Cells were centrifuged and stained at 4° C. inFACS buffer for 30 minutes containing FcR Blocking Reagent (MiltenyiBiotec), anti-CD19, anti-CD20, anti-CD40, anti-HLADR and anti-CD80.Cells were centrifuged and washed twice in FACS buffer and fixed in 0.5%paraformaldehyde. Cells were analyzed on Attune NxT Flow Cytometer(Thermo Fisher), with subsequent data analysis by Flowjo 10.7(Treestar). Dead cells were excluded by gating on the eFluor780-negative population. B cells were identified as CD19+CD20+ cells andlevel of activation marker was assessed by median fluorescent intensity.

For Ramos NFkb Reporter Assay, Ramos-Blue Cells NF-kB/AP-1 Reporter Blymphocytes were purchased from Invivogen. Cells were grown andmaintained in complete DMEM supplemented with 2 mM L-glutamine, 10% FBS,100 ug/mL Normacin, Pen-Strep, 100 ug/mL Zeocin. Stimulation of theRamos-Blue cells was performed. Briefly, cells were rinsed in growthmedium without antibiotics. Cells were counted and resuspended in freshcomplete DMEM without selection antibiotics at a density of 2×10⁶cell/mL. 20 uL of 10 uM CpG 7-7, CpG 12070 and ODN2006 titrated 1:5 wereadded to a flat-bottom 96-well plate, 180 uL of the cell suspension wereadded to a final concentration of 1 uM to 64 μM of CpG. Plate wasincubated at 37° C. in a 5% CO2 incubator for 24h. On day of assay, QBreagent and QB buffer were thawed before us. Quanti-Blue solution wasprepared by adding 1 mL of QB reagent and 1 mL of 1 mL of QB buffer to98 mL of sterile water in a sterile glass bottle. 180 uL of Quanti-Bluesolution was dispensed per well into a new flat-bottom 96-well plate. 20uL of supernatant from treated Ramos-Blue cells was then added to the96-well plate. Plate was ten incubated for 6h. Optical density wasmeasured at OD655 using a plate reader (Molecular Devices), and data wastabulated in GraphPad Prism 9.0.

Results

Human PBMCs were treated with free CpGs to evaluate their respectiveactivities as observed by CD40 expression on CD19 positive B cells. Asshown in FIG. 27 , series 7 CpGs (SEQ ID NOS: 29, 30, and 32-36) allshowed enhanced activities compared with CpG 12070 (SEQ ID NO: 3).

CpG oligos 7-7, 12070 and ODN2006 (5′-tcgtcgttttgtcgttttgtcgtt-3′; SEQID NO:167) were compared in a NFkb reporter assay. As shown in FIG. 28 ,CpG 7-7 showed significantly higher activity as compared to 12070 andODN2006.

Example 17: Evaluation of CpG Activity on PBMCs from Different Donors

The activity of CpG oligos 7-6, 7-7 and 12070 were compared for activityin PBMC cells from three different donor lines (D559, D804 and D643) asobserved by CD40 expression. The evaluation of activity of the CpGoligos was performed using the same methods as Example 16 above.

The results showed that the higher activities of 7-6 and 7-7 comparedwith 12070 were not dependent on the donor (FIGS. 29A-29C).

Example 18: Contributions of 5′ Bromo 2′Deoxyuridine and PEG Linkage toCpG Activity

For evaluation of CpG oligonucleotides in human PBMCs, Trima residualswere received from Vitalant and diluted 1:4 with Phosphate BufferedSaline (PBS, Gibco). Diluted blood was split into two tubes andunderplayed with 15 mL Ficoll-Paque (GE Healthcare). Tubes werecentrifuged for 30 minutes at 400×g. PBMCs were collected from theinterface and resuspended in FACS buffer (PBS with 0.5% Bovine SerumAlbumin (Gibco)). PBMCs were immediately plated onto a 96-well format(500K/well) in Complete RPMI (RPMI+10% FBS). Five-fold serial dilutionswere added to the cells from 1 uM to 64 μM of CpG polynucleotides at 37°C. under 5% CO2 for 48 to 96 hours. Cells were pelleted bycentrifugation for five minutes at 400×g and stained at 4° C. in FixableViability Dye eFluor 780 (Thermo Fisher) diluted 1:4000 in PBS. Cellswere centrifuged and stained at 4° C. in FACS buffer for 30 minutescontaining FcR. Blocking Reagent (Miltenyi Biotec), anti-CD19,anti-CD40, and anti-CD86. Cells were centrifuged and washed twice inFACS buffer and fixed in 0.5% paraformaldehyde. Cells were analyzed onAttune NxT Flow Cytometer (Thermo Fisher), with subsequent data analysisby Flowjo 10.7 (Treestar). Dead cells were excluded by gating on theeFluor 780-negative population. Gating CD19+, CD20+ or CD19+CD20+ cellsto identify B cells. Data was tabulated using GraphPad Prism 9.0.

As shown in FIG. 30 , CpG oligonucleotides 9-9 and 9-10 without thebromo modification at the 5′ uridine activated CD86 expression. Thisimplies that the bromo modification is not an essential component of therespective oligonucleotides.

Example 19: Biological Evaluation of CpG-Nucleotides and Antibody-CpGNucleotide Conjugates

Trima residuals were received from Vitalant and diluted 1:2 withPhosphate Buffered Saline (PBS, Gibco). Diluted blood was split into twotubes and underplayed with 15 mL Ficoll-Paque (GE Healthcare). Tubeswere centrifuged for 30 minutes at 400×g. PBMCs were collected from theinterface, resuspended and washed in FACS buffer (PBS with 0.5% BovineSerum Albumin (Gibco)). After one wash, PBMCs were resuspended inComplete RPMI (RPMI+10% FBS). PBMCs were immediately plated onto a96-well format (500K/well) in Complete RPMI. Five-fold serial dilutionswere added to the cells from 100 nM to 6.4 μM conjugated antibody at 37°C. under 5% CO2 for 48 hours. Cells were pelleted by centrifugation forfive minutes at 400×g and stained at 4° C. in Fixable Viability DyeeFluor 780 (Thermo Fisher) diluted 1:4000 in PBS. Cells were centrifugedand stained at 4° C. in FACS buffer for 30 minutes containing FcRBlocking Reagent (Miltenyi Biotec), anti-CD19, anti-CD20, anti-CD40,anti-HLADR and anti-CD80. Cells were centrifuged and washed twice inFACS buffer and fixed in 0.5% paraformaldehyde. Cells were analyzed onAttune NxT Flow Cytometer (Thermo Fisher), with subsequent data analysisby Flowjo 10.7 (Treestar). Dead cells were excluded by gating on theeFluor 780-negative population. B cells were identified as CD19+CD20+cells and level of activation marker was assessed by median fluorescentintensity.

TNT127=RL1_hKappa N92A+RH2_hIgG1_AAA+S-tag (SEQ ID Nos: 73 and 65,respectively)TNT130a=TNT127 with N92A mutation conjugated to CpG 12070 (DAR1)TNT133a=TNT127 with N92A mutation conjugated to CpG 7-6 (DAR1)TNT134a=TNT127 with N92A mutation conjugated to CpG 7-7 (DAR1)

The activity of antibody TNT127 was compared when conjugated to CpGs7-6, 7-7 and 12070. As shown in FIG. 31 , antibody conjugates with CpG7-6 (SEQ ID NO:34) and 7-7 (SEQ ID NO:35) demonstrated substantiallybetter activity as compared to the 12070 (SEQ ID NO:3) conjugate, asobserved by CD40 expression.

Example 20: Comparison of Anti-CD22 Antibody:CpG OligonucleotideConjugates

The activity of CD22 antibody-CpG conjugates DAR1 and DAR2 were comparedfor activity. In all cases tested, at the lower concentration ranges ofconjugate, the DAR2 conjugates displayed higher activity as compared tothe DAR1 conjugates (FIGS. 32A-32C). However, the DAR1 conjugates withthe 7-6 and 7-7 CpGs displayed higher activity that was more comparableto DAR2. Methods were as described in Example 19.

TNT135b=TNT127 with N92A mutation conjugated to CpG 12070 (DAR2)TNT136b=TNT127 with N92A mutation conjugated to CpG 7-6 (DAR2)TNT137b=TNT127 with N92A mutation conjugated to CpG 7-7 (DAR2)TNT130a=TNT127 with N92A mutation conjugated to CpG 12070 (DAR1)TNT133a=TNT127 with N92A mutation conjugated to CpG 7-6 (DAR1)TNT134a=TNT127 with N92A mutation conjugated to CpG 7-7 (DAR1)

Example 21: Anti-CD22 Antibody:CpG Oligonucleotide Conjugate MediatesTumor Killing in the Absence of an Active Fc Domain

Immune cell mediated anti-tumor response was tested in a tumor bearingsyngeneic model using anti-CD22 CpG oligonucleotide comprising aninactive Fc using the mouse IgG1 N297A Fc. CT26 mouse colon carcinomacells (ATCC) were cultured in Complete RPMI 1640 (RPMI1640+10% FBS(Gibco)) at 37° C. and 5% CO2. Once cells were 80% confluent, cells weredetached with Trypsin 0.25% (Gibco) and washed twice with RPMI 1640(Gibco). Cells were resuspended at 20E6/mL in RPMI 1640 and kept on iceuntil use. 100 uL of suspended cells were subcutaneously implanted intothe right flank of 6 week old female BALB/c mice (Charles River). Tumorsize was measured and recorded twice a week with calipers starting 7days post implantation until duration of the study, approximately 27days later. Tumor volume was estimated using the following formula:(length×width×width)/2. Once tumors reached 120 or 250 mm3,approximately 7 days post implantation, mice were randomized by tumorsize and treatments were initiated. The conjugates were administeredintravenously 1 to 3 doses every 3 days at 10 mg/kg. Mice whose tumorsexceeded 2,000 mm³ or exhibited any signs of distress at any time duringthe study were killed humanely as per IACUC-approved animal protocols.

As shown in FIG. 33 , antitumor activity was observed with all dosingschedules with most effective antitumor activity when the anti-CD22-4523conjugates (inactive Fc) being administered intravenously 1 to 3 dosesevery 3 days at 10 mg/kg.

Example 22: Activation of TLR9 by Anti-CD22 Antibody:CpG OligonucleotideConjugates

For biological evaluation of CpG-oligonucleotides and antibody-CpGoligonucleotide conjugates, Trima residuals were received from Vitalantand diluted 1:2 with Phosphate Buffered Saline (PBS, Gibco). Dilutedblood was split into two tubes and underplayed with 15 mL Ficoll-Paque(GE Healthcare). Tubes were centrifuged for 30 minutes at 400×g. PBMCswere collected from the interface, resuspended and washed in FACS buffer(PBS with 0.5% Bovine Serum Albumin (Gibco)). After one wash, PBMCs wereresuspended in Complete RPMI (RPMI+10% FBS). PBMCs were immediatelyplated onto a 96-well format (500K/well) in Complete RPMI. Five-foldserial dilutions were added to the cells from 1 uM to 64 μM of CpGpolynucleotides or 100 nM to 6.4 μM conjugated antibody at 37° C. under5% CO2 for 18 hours. Cells were pelleted by centrifugation for fiveminutes at 400×g and stained at 4° C. in Fixable Viability Dye eFluor780 (Thermo Fisher) diluted 1:4000 in PBS. Cells were centrifuged andstained at 4° C. in FACS buffer for 30 minutes containing FcR BlockingReagent (Miltenyi Biotec), anti-CD19, anti-CD20, anti-CD40, anti-HLADR,anti-CD80, anti-CD86, anti-CD3, anti-CD14, anti-CD11c, anti-CD69 andanti-CD56. Cells were centrifuged and washed twice in FACS buffer andfixed in 0.5% paraformaldehyde. Cells were analyzed on Attune NxT FlowCytometer (Thermo Fisher), with subsequent data analysis by Flowjo 10.7(Treestar). Dead cells were excluded by gating on the eFluor780-negative population. B cells were identified as CD19+CD20+ cells, Tcells as CD3+CD56−, DC as CD11chiHLADR+, and monocytes as CD14+ andlevel of activation marker was assessed by median fluorescent intensity.

TNT138a=anti-CD22 antibody having RH2 VH domain and RL1 N92S VL domain(SEQ ID Nos:65 and 87, respectively) conjugated to compound 7.7b (7-7)CpG (SEQ ID NO:35)

CpG (7-7b; SEQ ID NO:35), CD22 antibody (unconjugated) and CD22antibody-CpG conjugate (TNT138a, as described above) were compared forTLR9 activation in both TLR9 positive and TLR9 negative immune cells byobserving CD40, CD80 and CD69 expression. The CD22-CpG conjugateactivated TLR9 positive/CD22 positive B cells, whereas CpG alone wasunable to activate these cells except at higher concentrations of oligo(FIG. 34A). The CD22-CpG conjugate did not activate TLR9 positive cellsthat lacked the CD22 targeted by the conjugate, demonstrating thespecificity of delivering the CpG oligo activating function to onlycells with the CD22 target using RFB4 antibody and conjugate (FIGS.34B-34D). Unconjugated CD22 did not exhibit activation in any TLR9positive immune cells. The CpG, antibody and conjugate did not activateTLR9 negative immune cells as demonstrated by the lack of activation ofCD69 in T cells.

Example 23: Biological Evaluation of Anti-Her2 Antibody:CpGOligonucleotide Conjugates

Materials and Methods

A mouse spleen coculture was produced using splenocytes derived from thespleens of either a Balbc syngeneic mouse (full immune system) or from aNODSCID immunocompromised mouse (no B, T, dysfunctional DC, NK) (bothfrom (Charles River), each cultured separately in presence of humanbreast tumor cell line SKBR3 (Her2+++) (ATCC). The cocultures wereincubated with CpG conjugates or control. Biological evaluation ofCpG-nucleotides and antibody-CpG conjugates in the co-culture assay byobserving activation of monocytes, macrophages and dendritic cells wasassessed as follows.

SKBR3 were non enzymatically detached with TryplE (Thermo Scientific)and resuspended at 1E6/mL in PBS (Gibco). 300 nM of CarboxyfluoresceinSuccinimidyl Ester (CFSE (Thermo Scientific)) was added for 20 minutesat 37° C. Excess dye were removed from cells by washing with CompleteRPMI (RPMI1640+10% Fetal Bovine Serum (Gibco)). CFSE labeled SKBR3 cellswere immediately plated onto a 96-well format (100 k/well) in CompleteRPMI.

Fresh spleens were harvested from mice and passed through a 70 um filter(Fisher Scientific) into a 50 mL tube (Falcon) using the rubber end of a3 mL plunger (Falcon). The dissociated spleen was resuspended in 15 mLof FACs buffer (PBS+0.5% Bovine Serum Albumin (Gibco) and centrifugedfor 10 minutes at 400×g. Red blood cells were removed from splenocytesby adding 1 mL of ACK lysis buffer (Gibco) for 2 minutes beforeneutralizing with 15 mL of FACs buffer. Cells were centrifuged for 10minutes at 400×g and passaged through a new 70 uM filter. Splenocyteswere resuspended in Complete RPMI and were immediately plated (1E6/well)at a 10:1 ratio with the CFSE labeled SKBR3 cells.

Five-fold serial dilutions were added to the cells from 100 nM to 6.4 μMof antibody and conjugated antibody and 1 uM to 64 μM of CpGpolynucleotides at 37° C. under 5% CO2 for 48 hours. Cells were pelletedby centrifugation for five minutes at 400×g and stained for 30 minutesat 4° C. in Fixable Viability Dye eFluor 780 (Thermo Fisher) diluted1:4000 in PBS. Cells were centrifuged and stained at room temperature inFACS buffer for 5 minutes with FcR Blocking Reagent (Biolegend) and thenan additional 40 minutes at 4° C. containing anti-CD45, anti-CD49b,anti-CD3, anti-B220, anti-CD11b, anti-MHCII, anti-CD86, anti-CD40,anti-GR1, anti-F480 and anti CD11c. Cells were centrifuged and washedtwice in FACS buffer and fixed in 0.5% paraformaldehyde. Cells wereanalyzed on Attune NxT Flow Cytometer (Thermo Fisher), with subsequentdata analysis by Flowjo 10.7 (Treestar). Dead cells were excluded bygating on the eFluor 780-negative population. Lineage specific cellswere first excluded (CD3, B220, CD49b) prior to gating CD11b+GR1midF480+cells to identify monocyte macrophages and F480-CD11c+MHCII+ cells toidentify DC cells.

Anti-huHer2 antibody (SEQ ID Nos:126-128) was tested in context of mIgG1or mIgG2a Fc domain, either unconjugated or conjugated to 4523 murineCpG oligonucleotide (SEQ ID NO:121).

Results

This co-culture model provides an assessment of the activity of thetested CpG conjugates in the presence of a broader immune model system.The results showed that CpG conjugates with mIgG2a Fc domain activatedmonocytes, macrophages and dendritic cells to a greater extent than theCpG conjugates with mIgG1 Fc domain using spleenocytes from BALBc mice(FIGS. 35A & 35B)

Example 24: Evaluation of CD22 Ab-CpG Conjugate in an Immune CheckpointInhibitor Refractory Model

EMT6 mouse mammary carcinoma cells (ATCC) were cultured in Complete RPMI1640 (RPMI1640+10% FBS (Gibco)) at 37° C. and 5% CO2. Cells weredetached with Trypsin 0.25% (Gibco) and washed twice with RPMI 1640(Gibco). Cells were resuspended at 20E6/mL in RPMI 1640 and kept on iceuntil use. 100 uL of suspended cells were subcutaneously implanted intothe right flank of 6 week old female BALB/c mice (Charles River). Tumorsize was measured and recorded twice a week with calipers starting 3days post implantation until duration of the study, approximately 35days later. Tumor volume was calculated using the following formula:(length×width×width)/2. Once tumors reached on average 75 mm³,approximately 3 days post implantation, mice were randomized by tumorsize and treatments were initiated. In this study, the mice were treatedwith TNT50a, an anti-mouse CD22 antibody (SEQ ID NO: 124 and SEQ ID NO:125) conjugated to mouse CpG 4523 (SEQ ID NO: 121) of DAR1configuration. TNT50a conjugates were administered intraperitoneally 2doses every 3, 5 or 7 days at 10 mg/kg. Mice whose tumors exceeded 2,000mm³ or exhibited any signs of distress at any time during the study weresacrificed humanely as per IACUC-approved animal protocols.

As shown in FIG. 36 , TNT50a showed anti-tumor efficacy in all threedosing regimens. EMT6 breast model is generally refractory to treatmentby anti-PD1 and anti-PD-L1 treatment. Therefore, the data shows thatanti-CD22 Ab-CpG conjugate is superior showing potent single agentactivity in checkpoint inhibitor refractory model.

Example 25: Intratumoral Administration

EMT6 mouse mammary carcinoma cells (ATCC) were cultured in Complete RPMI1640 (RPMI1640+10% FBS (Gibco)) at 37° C. and 5% CO2. Cells weredetached with Trypsin 0.25% (Gibco) and washed twice with RPMI 1640(Gibco). Cells were resuspended at 20E6/mL in RPMI 1640 and kept on iceuntil use. 100 uL of suspended cells were subcutaneously implanted intothe right flank of 6 week old female BALB/c mice (Charles River). Tumorsize was measured and recorded twice a week with calipers starting 7days post implantation until duration of the study, approximately 25days later. Tumor volume was calculated using the following formula:(length×width×width)/2. Once tumors reached on average 180 mm³,approximately 7 days post implantation, mice were randomized by tumorsize and treatments were initiated. In this study, the mice were treatedwith TNT50a, an anti-mouse CD22 antibody (SEQ ID NO: 124 and SEQ ID NO:125) conjugated to mouse CpG 4523 (SEQ ID NO: 121) of DAR1configuration. TNT50a conjugates were administered intratumorally 2doses every 3 days at 24 uM. Mice whose tumors exceeded 2,000 mm³ orexhibited any signs of distress at any time during the study weresacrificed humanely as per IACUC-approved animal protocols.

As shown in FIG. 37 , the CD22 Ab-CpG conjugate showed anti-tumorpotency as did the free CpG (CpG-4253) when administered intratumorally.

Example 26: Immunophenotyping

Immunophenotyping of multiple syngeneic models administered with TNT50awere conducted. TNT50a, an anti-mouse CD22 antibody (SEQ ID NO: 124 andSEQ ID NO: 125) conjugated to mouse CpG 4523 (SEQ ID NO: 121) of DAR1configuration. A20 B cell lymphoma, CT26 colon carcinoma, EMT6 mousemammary carcinoma cells (ATCC) were cultured in Complete RPMI 1640(RPMI1640+10% FBS (Gibco)) at 37° C. and 5% CO2. Cells were detachedwith Trypsin 0.25% (Gibco) and washed twice with RPMI 1640 (Gibco).Cells were resuspended at 20E6/mL in RPMI 1640 and kept on ice untiluse. 100 uL of suspended cells were subcutaneously implanted into theright flank of 6 week old female BALB/c mice (Charles River). Tumor sizewas measured with calipers. Tumor volume was calculated using thefollowing formula: (length×width×width)/2. Once tumors reached onaverage 200-300 mm³, mice were randomized by tumor size and treatmentswere initiated. The conjugates were administered 1 or 2 dosesintraperitoneally every 3 days at 10 mg/kg. Mice whose tumors exceeded2,000 mm³ or exhibited any signs of distress at any time during thestudy were sacrificed humanely as per IACUC-approved animal protocols.

Spleens and tumors were harvested either two or three days post-lastinjection for immunophenotyping. Spleens were processed into single-cellsuspension in ice-cold PBS, lysed with ACK lysis buffer (Gibco), washedtwice and re-suspended in PBS supplemented with 2% FBS. Tumor-derivedsingle-cell suspensions were prepared using a cocktail of Collagenase A(Roche), Collagenase D (Roche) and DNAse (Roche) for 45 min at 37° C.Cell counts were performed using ViCell counter (Beckman Coulter) forspleen and trypan blue exclusion with hemacytometer for tumor. Aliquotsof 1-2×10⁶ cells were either used for cell-surface antigen staining orstimulation for cytokine assessment. For surface staining, cells werestained with LIVE/DEAD fixable dye (Thermo Fisher), followed by mouseFc-block (Bio-legend) and subsequently stained with antibodies accordingto cell-type specific antibody panels for at least 30 min at 4° C.Following antibodies were used: IgD, CD19, CD95, CD3, CD11b, IL-10,Cd1d, CD5, CD138, CD44, CD4, CD8, CD45, CD62L, IFNg, TNFa, CD25, FOXP3,KI67, CD11c, MHCII, Ly6C, CD64. All flow antibodies were purchased fromeither Biolegend or Thermofisher.

As shown in FIGS. 38-40 , the CD22 Ab-CpG conjugate promotes B celldifferentiation, decrease in Bregs, increase in T cell effector functionand modulation of suppressive myeloid cells. FIGS. 38A-38D show theincrease in B cell differentiation and decrease in B regulatory cells inthe spleen following administration of the CD22 Ab-CpG conjugate. FIGS.39A-39D show the increase in CD4 and CD8 T effector cells and functionin the spleen following administration of the CD22 Ab-CpG conjugate.FIGS. 40A-40D show the increase in B cell infiltrates and modulation ofsuppressive microenvironment in the tumor following administration ofthe CD22 Ab-CpG conjugate.

Example 27: Biological Evaluation of Human B Cell Derived CytokineExpression

Trima residuals were received from Vitalant and diluted 1:2 withPhosphate Buffered Saline (PBS, Gibco). Diluted blood was split into twotubes and underplayed with 15 mL Ficoll-Paque (GE Healthcare). Tubeswere centrifuged for 30 minutes at 400×g. PBMCs were collected from theinterface, resuspended and washed in FACS buffer (PBS with 0.5% BovineSerum Albumin (Gibco)). After one wash, PBMCs were resuspended inComplete RPMI (RPMI+10% FBS). PBMCs were immediately plated onto a96-well format (1e6/well) in Complete RPMI. Five-fold serial dilutionswere added to the cells from 1 uM to 64 μM of CpG polynucleotides at 37°C. under 5% CO2 for 48 hours. Cells were pelleted by centrifugation forfive minutes at 400×g and stained at 4° C. in Fixable Viability DyeeFluor 780 (Thermo Fisher) diluted 1:4000 in PBS. Cells were centrifugedand stained at 4° C. in FACS buffer for 30 minutes containing FcRBlocking Reagent (Miltenyi Biotec), anti-CD19, anti-CD40, anti-CD86,anti-CD25. Cells were centrifuged and washed twice in FACS buffer. Cellswere then processed for intracellular staining using the Transcriptionfactor fixation/permeabilization concentrate and diluent (eBioscience).

Briefly, cells were incubated in fresh fixation buffer by mixing 1 partof fixation/permeabilization concentrate with 3 parts of fixationpermeabilization diluent. Samples were incubated for 30-60 min at 4° C.protected from light. Samples were then centrifuged at 600 g for 5 minat room temperature. Resuspended pellet with 1× permeabilization bufferfollowed by two rounds of washes and centrifugation at 600g for 5 min atroom temperature. Pellets were resuspended in 100 uL of permeabilizationbuffer and stained with anti-CCL3, anti-IL2 and anti-IL6 for 60 min atroom temperature. Cells were centrifuged and washed twice in FACS bufferand fixed in 0.5% paraformaldehyde. Cells were analyzed on Attune NxTFlow Cytometer (Thermo Fisher), with subsequent data analysis by Flowjo10.7 (Treestar). Dead cells were excluded by gating on the eFluor780-negative population. B cells were identified as CD19+ cells, levelsof activation marker were assessed by median fluorescent intensity andcytokine/chemokine expression was assessed as a % of CD19+ cells. Inthese experiments, unconjugated anti-human CD22 antibody (SEQ ID NO: 65and SEQ ID NO: 87), free CpG (SEQ ID NO: 35, 7-7b) and anti-human CD22antibody (SEQ ID NO: 65 and SEQ ID NO: 87) conjugated to CpG 7-7 (SEQ IDNO: 35) were tested.

FIGS. 41A-41C show robust induction of B cell cytokines and chemokinesupon CD22− mediated TLR9 engagement in human B cells.

Example 28: Biological Evaluation of Murine Serum Cytokine LevelsFollowing Repeat Dosing with Anti-mCD22-CpG Conjugate

CT26 colon carcinoma cells (ATCC) were cultured in Complete RPMI 1640(RPMI1640+10% FBS (Gibco)) at 37° C. and 5% CO2. Cells were detachedwith Trypsin 0.25% (Gibco) and washed twice with RPMI 1640 (Gibco).Cells were resuspended at 20E6/mL in RPMI 1640 and kept on ice untiluse. 100 uL of suspended cells were subcutaneously implanted into theright flank of 6 week old female BALB/c mice (Charles River). Whentumors reached an average of 300 mm³, mice were randomized into twogroups and dosed intraperitoneally with 10 mg/kg of TNT50a,anti-mCD22-CpG or PBS twice, 3 days apart. Forty-eight hours post-eachdose mice, and 9 days post-last dose were bled into serum microtainertubes (BD). Serum was collected following centrifugation and stored at-80C for cytokine evaluation using Isoplexis platform. Briefly, serumsamples were thawed at room temperature along with Mouse CodePlexSeretome chip (Isoplexis). Serum samples were loaded neat in duplicatesinto chip chambers. Following the loading of a calibration chip, theCodeplex Secretome chip was loaded into the Isolight for analysis.Sensitivity range for this assay is 5-5000 pg/mL for 16 evaluatedcytokines and chemokines. The anti-mCD22 conjugate used in this study,TNT50a, is an anti-mouse CD22 antibody (SEQ ID NO: 124 and SEQ ID NO:125) conjugated to mouse CpG 4523 (SEQ ID NO: 121).

FIGS. 42A & 42B show robust induction of various effector cytokines andchemokines with anti-mCD22-CpG with no apparent accumulation in theperiphery upon repeat dosing in mouse.

Example 29: Tumor Killing in PD1 Non-Responders

Immune cell mediated anti-tumor response was tested in a tumor bearingsyngeneic model using TNT50a, which is an anti-mouse CD22 antibody (SEQID NO: 124 and SEQ ID NO: 125) conjugated to mouse CpG 4523 (SEQ ID NO:121). CT26 mouse colon carcinoma cells (ATCC) were cultured in CompleteRPMI 1640 (RPMI1640+10% FBS (Gibco)) at 37° C. and 5% CO2. Once cellswere 80% confluent, cells were detached with Trypsin 0.25% (Gibco) andwashed twice with RPMI 1640 (Gibco). Cells were resuspended at 20E6/mLin RPMI 1640 and kept on ice until use. 100 uL of suspended cells weresubcutaneously implanted into the right flank of 6 week old femaleBALB/c mice (Charles River). Tumor size was measured and recorded twicea week with calipers starting 5 days post implantation until duration ofthe study, approximately 30 days later. Tumor volume was estimated usingthe following formula: (length×width×width)/2. Once tumors reached50-200 mm³, approximately 5 days post implantation, mice were randomizedby tumor size and treatment with anti-PD1 (clone RPMI14 BioXCell) andPBS was initiated. Anti-PD1 and PBS (Gibco) were administeredintraperitoneally for 2 doses every 3 days at 10 mg/kg and 3 doses every3 days respectively. Approximately 11 days post implantation, anti-PD1treated mice were measured and re-randomized by tumor size. Mice whosetumor progressed from initial size and measured greater than 200 mm³were considered anti-PD1 non responders. Mice within the anti-PD1 nonresponder group was re-randomized by tumor size and conjugate treatmentwas initiated. The conjugates were administered intravenously for 2doses every 3 days at 10 mg/kg. For combination and single arm control,anti-PD1 treatment continued by intraperitoneal dosing every 3 days at10 mg/kg for 2 doses. Mice whose tumors exceeded 2,000 mm³ or exhibitedany signs of distress at any time during the study were sacrificedhumanely as per IACUC-approved animal protocols. FIG. 43 shows thattumor volume was reduced in mice treated with conjugate TNT50a, and micetreated with a combination of conjugate TNT50a and anti-PD1 antibody,whereas mice treated with only anti-PD1 antibody did not experience asignificant reduction in tumor volume.

Example 30: Reduction of Lung Metastatic Burden

BALB/C mice were implanted with 4T1 cells, treatment with TNT50ainitiated around day 5 when the tumor is around 60 mm³ at 10 mg/kg, 3doses every 3 days. TNT50a, is an anti-mouse CD22 antibody (SEQ ID NO:124 and SEQ ID NO: 125) conjugated to mouse CpG 4523 (SEQ ID NO: 121)with a DAR1 configuration. The lungs were harvested 8-9 days post-lastinjection for metastatic nodule quantification. In brief, lungs wereharvested in ice-cold 1×PBS, minced into small pieces then transferredinto digestion solution consisting of 2 mg/mL collagenase type V(Worthington) supplemented with 50 ug/mL DNAse (Sigma) and incubated for2 hrs in a 37C incubator with end-over-end rotation. Suspension wastransferred into 70 um strainer, washed once in 1×PBS then transferredinto 10 mL selection media consisting of RMPI 1640 supplemented with 10%FBS, penicillin-streptomycin and 10 ug/mL 6-thioguanine. Three to four1:10 serial dilutions were plated either in 6 well plates or 10 cmdishes and cultured for 10-14 days at 37C, 5% CO2. Metastatic plaqueswere then fixed in methanol for 5 min at room temperature, re-hydratedin distilled water then stained with 0.03% methylene blue for 5 min atroom temperature. Dye was then discarded, plate was rinsed gently withdistilled water and allowed to air-dry prior to counting plaques.

As shown in FIG. 44 , the number of metastatic plaques was significantlyreduced in the CD22 Ab-CpG conjugate as compared to the control.

1. A conjugate comprising an antibody or antigen-binding fragmentthereof and one or more immunomodulating oligonucleotides (P), whereinthe antibody or antigen-binding fragment is linked to one or more Q-tagpeptides (Q) comprising at least one glutamine residue, and wherein eachimmunomodulating oligonucleotide is linked to a Q-tag peptide via anamide bond with the glutamine residue of the Q-tag peptide and a linker(L) as shown in Formula (A):

wherein

indicates the point of attachment of each Q to the antibody orantigen-binding fragment thereof (Ab); wherein each P is independentlyan immunomodulating oligonucleotide having the structure

wherein

and

indicate the points of attachment within the oligonucleotide, andwherein

indicates the point of attachment to the linker L; each T¹ isindependently O or S; each T₂ is S⁻; T³ is a group

 wherein

indicates the point of attachment to L and wherein,

indicates the point of attachment to the rest of the oligonucleotide; Zis O or S; U^(5′) is —H or halogen; R^(5′) is —H or methoxy; R is —H ormethoxy; R^(g1), R^(g2), R^(g3), and R^(g4) are H; R^(3′) is methoxy; R¹is —(CH₂)₃—OH; R² is —H or methyl; and n is an integer from 0 to 2.2-287. (canceled)
 288. The conjugate of claim 1, wherein at least oneQ-tag peptide sequence is selected from the group consisting of SEQ IDNOs: 40-55.
 289. The conjugate of claim 1, wherein the antibody orfragment thereof specifically binds human CD22.
 290. The conjugate ofclaim 1, wherein the antibody comprises a heavy chain variable (VH)domain and a light chain variable (VL) domain, wherein: (a) the VHdomain comprises CDR-H1, CDR-H2, and CDR-H3 sequences from a VH domainsequence selected from the group consisting of SEQ ID Nos:64-67; (b) theVH domain comprises a CDR-H1 comprising the sequence of SEQ ID NO:113, aCDR-H2 comprising the sequence of SEQ ID NO:115, and a CDR-H3 comprisingthe sequence of SEQ ID NO:116; or (c) the VH domain comprises a CDR-H1comprising the sequence of SEQ ID NO:114, a CDR-H2 comprising thesequence of SEQ ID NO:189, and a CDR-H3 comprising the sequence of SEQID NO:116; or (d) the VH domain comprises an amino acid sequenceselected from the group consisting of SEQ ID Nos:64-67.
 291. Theconjugate of claim 1, wherein the antibody comprises a heavy chainvariable (VH) domain and a light chain variable (VL) domain, wherein:(a) the VL domain comprises CDR-L1, CDR-L2, and CDR-L3 sequences from aVL domain sequence selected from the group consisting of SEQ IDNos:68-91; (b) the VL domain comprises a CDR-L1 comprising the sequenceof SEQ ID NO:117, a CDR-L2 comprising the sequence of SEQ ID NO:119, anda CDR-L3 comprising the sequence of SEQ ID NO:120; (c) the VL domaincomprises a CDR-L1 comprising the sequence of SEQ ID NO:118, a CDR-L2comprising the sequence of SEQ ID NO:177, and a CDR-L3 comprising thesequence of SEQ ID NO:120; or (d) the VL domain comprises an amino acidsequence selected from the group consisting of SEQ ID Nos:68-91. 292.The conjugate of claim 291, wherein the VL domain further comprises anamino acid substitution at residue N92 (numbering starting at theN-terminus of the VL domain sequence) selected from the group consistingof N92A, N92L and N92S.
 293. The conjugate of claim 1, wherein theantibody comprises a human Fc region selected from the group consistingof an IgG1 Fc region, an IgG2 Fc region, and an IgG4 Fc region.
 294. Theconjugate of claim 293, wherein the Fc region is: (a) a human IgG1 Fcregion comprising L234A, L235A, and/or G237A substitutions, amino acidposition numbering according to EU index; (b) a human IgG2 Fc regioncomprising A330S and/or P331S substitutions, amino acid positionnumbering according to EU index; or (c) a human IgG4 Fc regioncomprising S228P and/or L235E substitutions, amino acid positionnumbering according to EU index.
 295. The conjugate of claim 294,wherein the Fc region further comprises an N297A substitution, aminoacid position numbering according to EU index.
 296. The conjugate ofclaim 1, wherein the antibody or fragment thereof specifically bindsHER2.
 297. A conjugate comprising a protein, at least one Q tag peptidesequence selected from the group consisting of SEQ ID NOs: 40-55, and atleast one immunomodulatory oligonucleotide (P), and wherein eachimmunomodulatory oligonucleotide is linked to a Q tag via an amide bondwith a glutamine residue in the Q tag.
 298. The conjugate of claim 297,wherein each immunomodulating oligonucleotide (P) is linked to a Q-tagpeptide via an amide bond with the glutamine residue of the Q-tagpeptide and a linker (L) as shown in Formula (A):

wherein

indicates the point of attachment of each Q to the protein.
 299. Theconjugate of claim 298, wherein the protein is an antibody or antibodyfragment.
 300. The conjugate of claim 298, wherein the antibodycomprises two antibody light chains, two antibody heavy chains, and twoQ-tag peptides comprising a peptide sequence RPQGF (SEQ ID NO:47);wherein each of the Q-tag peptides is linked to the C-terminus of one ofthe antibody heavy chains; and wherein at least one of the Q-tagpeptides is linked to the immunomodulating oligonucleotide (P) via anamide bond with the glutamine residue of the Q-tag peptide and linker(L).
 301. The conjugate of claim 298, wherein each L is independently abond or a linker moiety

wherein m is an integer ranging from about 0 to about 50, and wherein

indicates the point of attachment to P, and

indicates the point of attachment to the rest of the conjugate connectedto Q via an amide bond with the glutamine residue; and each P isindependently an immunomodulating oligonucleotide having the structure

wherein

and

indicate the points of attachment within the oligonucleotide, andwherein

indicates the point of attachment to the linker L; each T¹ isindependently O or S; each T² is S⁻; provided that each P comprises oneor two pairs of geminal T¹ and T² wherein T¹ is S and T² is S, and theone or two pairs of geminal T¹ and T² are between nucleoside residues 2and 3, between nucleoside residues 3 and 4, between nucleoside residues5 and 6, between nucleoside residues 6 and 7, between nucleosideresidues 7 and 8, between nucleoside residues 8 and 9, betweennucleoside residues 9 and 10, or between nucleoside residues 10 and 11;T³ is a group

wherein

indicates the point of attachment to L and wherein

indicates the point of attachment to the rest of the oligonucleotide; Zis O or S; U^(5′) is —H or halogen; R^(5′) is —H; R^(c1) is —H; R^(g1),R^(g2), R^(g3), and R^(g4) are H; R^(3′) is methoxy; R¹ is —(CH₂)₃—OH;R² is -methyl; and n is
 1. 302. The conjugate of claim 301, wherein eachP is independently an immunomodulating oligonucleotide having thestructure

or having the structure

wherein

and

indicate the points of attachment within the oligonucleotide, andwherein

indicates the point of attachment to the linker L; and wherein theprotein is an antibody comprising a heavy chain variable (VH) domain anda light chain variable (VL) domain.
 303. The conjugate of claim 302,wherein: (a) the VH domain comprises a CDR-H1 comprising the sequence ofSEQ ID NO:113, a CDR-H2 comprising the sequence of SEQ ID NO:115, and aCDR-H3 comprising the sequence of SEQ ID NO:116, and the VL domaincomprises a CDR-L1 comprising the sequence of SEQ ID NO:117, a CDR-L2comprising the sequence of SEQ ID NO:119, and a CDR-L3 comprising thesequence of SEQ ID NO:120; (b) the VH domain comprises a CDR-H1comprising the sequence of SEQ ID NO:114, a CDR-H2 comprising thesequence of SEQ ID NO:189, and a CDR-H3 comprising the sequence of SEQID NO:116, and the VL domain comprises a CDR-L1 comprising the sequenceof SEQ ID NO:117, a CDR-L2 comprising the sequence of SEQ ID NO:119, anda CDR-L3 comprising the sequence of SEQ ID NO:120; (c) the VH domaincomprises a CDR-H1 comprising the sequence of SEQ ID NO:113, a CDR-H2comprising the sequence of SEQ ID NO:115, and a CDR-H3 comprising thesequence of SEQ ID NO:116, and the VL domain comprises a CDR-L1comprising the sequence of SEQ ID NO:118, a CDR-L2 comprising thesequence of SEQ ID NO:177, and a CDR-L3 comprising the sequence of SEQID NO:120; or (d) the VH domain comprises a CDR-H1 comprising thesequence of SEQ ID NO:114, a CDR-H2 comprising the sequence of SEQ IDNO:189, and a CDR-H3 comprising the sequence of SEQ ID NO:116, and theVL domain comprises a CDR-L1 comprising the sequence of SEQ ID NO:118, aCDR-L2 comprising the sequence of SEQ ID NO:177, and a CDR-L3 comprisingthe sequence of SEQ ID NO:120.
 304. The conjugate of claim 302, wherein:(a) the antibody comprises a heavy chain comprising the sequence of SEQID NO:179 and a light chain comprising the sequence of SEQ ID NO:181;(b) the antibody comprises a heavy chain comprising the sequence of SEQID NO:180 and a light chain comprising the sequence of SEQ ID NO:181;(c) the antibody comprises a heavy chain comprising the sequence of SEQID NO:179 and a light chain comprising the sequence of SEQ ID NO:182; or(d) the antibody comprises a heavy chain comprising the sequence of SEQID NO:180 and a light chain comprising the sequence of SEQ ID NO:182.305. An immunomodulating oligonucleotide of Formula (C):

wherein

and

indicate the points of attachment within the oligonucleotide; each T¹ isindependently O or S; each T² is S⁻; T³ is a group

 wherein m is an integer from 0 to 50 and wherein

indicates the point of attachment to the rest of the oligonucleotide; Zis O or S; U^(5′) is —H or halogen; R^(5′) is —H or methoxy; R^(c1) is—H or methoxy; R^(g1), R^(g2), R^(g3), and R^(g4) are H; R^(3′) ismethoxy; R¹ is —(CH₂)₃—OH; R² is —H or methyl; and n is an integer from0 to 2, or a pharmaceutically acceptable salt thereof.
 306. Animmunomodulating oligonucleotide of formula (D):

wherein

and

indicate the points of attachment within the oligonucleotide; each T¹ isindependently O or S; each T² is S⁻; T³ is a group

 wherein

indicates the point of attachment to L and wherein

indicates the point of attachment to the rest of the oligonucleotide; Lis a group

 wherein m is an integer from 0 to 50 and wherein

indicates the point of attachment to the rest of the oligonucleotide viaT³; Z is O or S; U^(5′) is —H or halogen; R^(5′) is —H or methoxy; R is—H or methoxy; R^(g1), R^(g2), R^(g3), and R^(g4) are H; R^(3′) ismethoxy; R¹ is —(CH₂)₃—OH; R² is —H or methyl; and n is an integer from0 to 2, or a pharmaceutically acceptable salt thereof.
 307. A method forpreparing a conjugate that comprises an antibody or antigen-bindingfragment thereof (Ab) and one or more immunomodulating oligonucleotides(P), wherein the antibody or antigen-binding fragment is linked to oneor more Q-tag peptides (Q), and wherein each immunomodulatingoligonucleotide is linked to a Q-tag peptide via an amide bond with theglutamine residue of the Q-tag peptide and a linker (L) as shown informula (A),

wherein:

indicates the point of attachment of each Q to the antibody orantigen-binding fragment thereof (Ab); each Q is independently a Q-tagpeptide having at least one glutamine residue; each L is independently abond or a linker moiety connected to Q via an amide bond with theglutamine residue; and each P is independently an immunomodulatingoligonucleotide; comprising contacting a compound of formula (B)

wherein Ab and Q are as defined for formula (A) above, and e is aninteger from 1 to 20, with one or more immunomodulating oligonucleotidesP, wherein each oligonucleotide P is independently an immunomodulatingoligonucleotide of formula (C) according to claim 305, in the presenceof a transglutaminase.
 308. A method for treating cancer, comprisingadministering to an individual an effective amount of a pharmaceuticalcomposition comprising the conjugate of claim
 1. 309. A method fortreating cancer, comprising administering to an individual an effectiveamount of a pharmaceutical composition comprising the conjugate of claim297.
 310. A method for treating cancer, comprising administering to anindividual an effective amount of a pharmaceutical compositioncomprising the immunomodulating oligonucleotide of claim
 305. 311. Amethod for treating cancer, comprising administering to an individual aneffective amount of a pharmaceutical composition comprising theimmunomodulating oligonucleotide of claim
 306. 312. An antibody thatbinds to human CD22, wherein the antibody comprises an antibody heavychain and an antibody light chain, wherein the antibody heavy chaincomprises the sequence of SEQ ID NO:179 or 180, and the antibody lightchain comprises the sequence of SEQ ID NO:181 or
 182. 313. An antibodythat binds to human Her2, wherein the antibody comprises an antibodyheavy chain and an antibody light chain, wherein the antibody heavychain comprises the sequence of SEQ ID NO:183, 184, 186, or 187, and theantibody light chain comprises the sequence of SEQ ID NO:185 or 188.